Treatment of balance impairments

ABSTRACT

Compositions and methods are provided for prophylactic or therapeutic treatment of balance impairments involving neuronal damage, loss, or degeneration, preferably of vestibular ganglion neurons, in an animal by administration of an effective amount of a trkB or trkC agonist, particularly a neurotrophin, more preferably NT-4/5

BACKGROUND FIELD OF THE INVENTION

[0001] This application relates to methods for prophylactic andtherapeutic treatment of balance impairments. More particularly, theapplication relates to prevention or therapy of ototoxin-induced balanceimpairments by administration of neurotrophins.

INTRODUCTION

[0002] Balance impairments are serious handicaps which affect millionsof people. Balance impairments can be attributed to a wide variety ofcauses, including infections, mechanical injury, loud sounds, aging, andchemical-induced ototoxicity that damage neurons and/or hair cells ofthe peripheral vestibular systems. Vestibular ganglion neurons (“VGN”),which are primary afferent sensory neurons responsible for balance,deliver signals from the utricle, saccule and ampullae of the inner earto the brain through the eighth nerve connecting primary auditoryneurons in the spiral ganglia to the brain stem. Damage to theperipheral auditory system is responsible for a majority of balancedeficits (Dublin, 1976; Lim, 1986) with destruction of vestibularganglia neurons as a major cause of balance impairments.

[0003] During embryogenesis, the vestibular ganglion, spiral ganglion,and the otic vesicle are derived from the same neurogenic ectoderm, theotic placode. The vestibular ganglion neurons send peripheral neuronalprojections to hair cells of the inner ear and extend centralprojections to the brainstem nuclei. This system is sensitive toototoxins that include therapeutic drugs, antineoplastic agents,contaminants in foods or medicines, and environmental and industrialpollutants. Ototoxic drugs include the widely used chemotherapeuticagent cisplatin and its analogs (Fleischman et al., 1975; Stadnicki etal., 1975; Nakai et al., 1982; Berggren et al., 1990), commonly usedaminoglycoside antibiotics, e.g. gentamicin, for the treatment ofinfections caused by Gram-negative bacteria, (Sera et al., 1987;Hinojosa and Lerner, 1987; Bareggi et al., 1990), quinine and itsanalogs, salicylate and its analogs, and loop-diuretics.

[0004] The toxic effects of these drugs on vestibular ganglion neuronsare often the limiting factor for their therapeutic usefulness. Forexample, antibacterial aminoglycosides such as gentamicins,streptomycins, kanamycins, tobramycins, and the like are known to haveserious toxicity, particularly ototoxicity and nephrotoxicity, whichreduce the usefulness of such antimicrobial agents (see Goodman andGilman's The Pharmacological Basis of Therapeutics, 6th ed., A. Goodmanand Gilman et al., eds; Macmillan Publishing Co., Inc., New York, pp.1169-71 (1980)). Aminoglycoside antibiotics are generally utilized asbroad spectrum antimicrobials effective against, for example,gram-positive, gram-negative and acid-fast bacteria. Susceptiblemicroorganisms include Escherichia spp., Haemohilus spp., Listeria spp.,Pseudomonas spp., Nocardia spp., Yersinia spp., Klebsiella spp.,Enterobacter spp., Salmonella spp., Staphyloccocus spp., Streptococcusspp., Mycobacteria spp., Shigella spp., and Serratia spp. Nonetheless,the aminoglycosides are used primarily to treat infections caused bygram-negative bacteria and, for instance, in combination withpenicillins for the synergistic effects. As implied by the generic namefor the family, all the aminoglycoside antibiotics contain aminosugarsin glycosidic linkage. Ototoxicity is a dose-limiting side-effect ofantibiotic administration. For example, nearly 75% of patients given 2grams of streptomycin daily for 60 to 120 days displayed some vestibularimpairment, whereas at 1 gram per day, the incidence decreased to 25%(U.S. Pat. No. 5,059,591). Ototoxicity is also a serious dose-limitingside-effect for cisplatin, a platinum coordination complex, that hasproven effective on a variety of human cancers including testicular,ovarian, bladder, and head and neck cancer. Cisplatin damages vestibularsystems (Fleischman et al., 1975; Stadnicki et al., 1975; Nakai et al.,1982; Carenza et al., 1986; Sera et al., 1987; Hinojosa and Lerner,1987; Bareggi et al., 1990).

[0005] Accordingly, there exists a need for means to prevent, reduce ortreat the incidence and/or severity of ototoxin-induced balanceimpairment related to vestibular neurons, particularly that arising asan unwanted side-effect of ototoxic therapeutic drugs, which includecisplatin and its analogs and aminoglycoside antibiotics. In addition,there exits a need for methods that allow higher and thus more effectivedosing with these ototoxicity-inducing balance-impairing therapeuticdrugs by concomitantly preventing or reducing the ototoxic effects ofthese drugs. What is needed is a method that provides a safe, effective,and prolonged means for prophylactic or curative treatment ofototoxin-induced balance impairment. In addition there is needed arapid, reliable, and facile system for testing the effects andmechanisms of ototoxins on balance in animals, including humans, and fortesting the efficacy of therapeutics to prevent, reduce or treat theseimpairments. The present invention provides such methods and systems toachieve these goals and others as well.

SUMMARY

[0006] The present invention results from the discovery disclosed hereinthat administration of certain neurotrophins can prevent or reducegentamicin- and cisplatin-induced cell death of vestibular ganglionneurons in dissociated cell culture in a dose-dependent manner. Whenneurotrophins or other growth factors were added together with cisplatinor gentamicin to a VGN culture, VGNs were specifically protected byneurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF) andneurotrophin-3 (NT-3), but not by NGF or other growth factors, includingepidermal growth factor (EGF), basic fibroblast growth factor (βFGF),and insulin-like growth factor-1 (IGF-1). It is one object of theinvention to provide a method for treating a mammal to prevent, reduce,or treat the incidence of or severity of an neuron-related balanceimpairment, particularly an ototoxin-induced or -inducible balanceimpairment, by administering to a mammal in need of such treatment atrkB or trkC agonist composition containing a prophylactically ortherapeutically effective amount of trkB or trkC agonist. The trkB ortrkC agonist is preferably a neurotrophin, more preferably NT-4/5, NT-3,or BDNF, and most preferably NT-4/5, or a functional fragment orderivative thereof, a chimeric neurotrophin, a pantropic neurotrophin,or a small molecule or antibody agonist thereof.

[0007] According to the method of this invention a composition of theinvention can be administered at a suitable interval(s) either prior to,subsequent to, or substantially concurrently with the administration ofor exposure to balance-impairment inducing neuronal damage, preferablyototoxin-induced or -inducible balance impairment. It is another objectof the invention to provide a method for treating a mammal to prevent,reduce, or treat neuronal-damage-related balance impairments, preferablyan ototoxin-induced balance impairment, by administering to a mammal inneed of such treatment a composition containing a prophylactically ortherapeutically effective amount of the trkB or trkC agonist incombination with a prophylactically or therapeutically effective amountof a second trkB or trkC agonist or an agent that acts synergisticallyor additively to enhance or complement the prophylactic or therapeuticeffect of the first trkB or trkC agonist.

[0008] It is another object of the invention to provide an improvedcomposition containing an ototoxicity-reducing or -preventing effectiveamount of the trkB or trkC agonist in combination with an ototoxicbalance-impairment inducing pharmaceutical drug for administration to amammal.

[0009] Such improved compositions can further contain a pharmaceuticallyacceptable carrier. The pharmaceutical composition will have lowerototoxicity than the ototoxic pharmaceutical alone, and preferably, havea higher dosage of the ototoxic pharmaceutical than typically used.Examples of such improved compositions include cisplatin or otherototoxic cancer agents or an aminoglycoside antibiotic(s) in combinationwith a trkB or trkC agonist.

[0010] Still further, the invention relates to the use in medicine ofcompositions of the invention in cases of bacterial infection. Thepresent invention provides a solution to the art that has long sought atherapy and a medicament which can prevent, reduce or treat the ototoxicbalance impairment effects currently associated with certainantibiotics, and particularly with the more popular and commonly usedaminoglycoside antibiotics without sacrificing the antimicrobialeffectiveness of the aminoglycosides.

[0011] Still further, the invention relates to the use in medicine ofcompositions of the invention in cases of cancer. The present inventionprovides a solution to the art that has long sought a therapy and amedicament which can prevent, reduce, or treat the ototoxic balanceimpairment effects currently associated with certain chemotherapeutics,and particularly with the more popular and commonly used cisplatinchemotherapeutics without sacrificing the antineoplastic effectivenessof cisplatin or its analogs.

[0012] Still further, the invention relates to the use in medicine ofcompositions of the invention in cases where diuretics are needed. Thepresent invention provides a solution to the art that has long sought atherapy and a medicament which can prevent, reduce, or treat theototoxic balance impairment effects currently associated with certaindiuretics, and particular with the more popular and commonly usedloop-diuretics, without sacrificing their diuretic effectiveness.

[0013] Finally, it is an object of the invention to provide aorganotypic utricle explant culture system that allows reliable, rapid,and facile determination of the ototoxic effect of compounds and theprophylactic or therapeutic effect of candidate compositions and methodsof the invention.

[0014] Additional objects and features of the invention will be apparentto those skilled in the art from the following detailed description andappended claims when taken in conjunction with the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a histogram depicting the effects of NT-4/5 on VGNsurvival. VGNs were prepared from P5 rats, plated and cultured for 2days in serum-free medium in the absence or presence of NT-4/5 atdifferent concentrations. Viable VGNs were identified by neurofilamentmonoclonal antibody (N52) labeling under a Zeiss Axiophoto microscopeand counted using a grid ocular reticule covering an area of 1 mm². Foreach culture, about 10 randomly selected fields were counted. Data werecollected from triplet cultures, and normalized as a percentage of thenumber of viable neurons in the control cultures in each of theexperiments. The error bars indicate SEM. As compared to controlcultures, NT-4/5 displayed very significant survival-promoting effectson VGNs at all doses tested (p<0.01, 2-tailed, unpaired t-test).

[0016]FIG. 2 is a histogram depicting the effects of neurotrophicfactors on VGN survival.

[0017] VGNs were prepared from P5 rats and kept for 2 days in serum-freemedium without or with different neurotrophins or other growth factorsat a concentration of 10 ng/ml. Quantitation of viable VGNs was done inthe same way as in FIG. 2 and the error bars represent SEM. Whencompared to control cultures, NT-3, BDNF and NT-4/5 all showed verysignificant survival promoting effects (p<0.001) In contrast, NGF, EGF,βFGF and IGF-1 did not produce significant effects (p >0.05). Thedifference in effectiveness between NT-3 and NT-4/5 or BDNF wassignificant (p<0.05).

[0018]FIG. 3 trkB-IgG and trkC-IgG inhibit the survival-promotingactivity of NT-4/5 or BDNF and NT-3, respectively. VGNs were preparedfrom P5 rats and exposed for 2 days to serum-free medium containing 1μg/ml trkB-IgG or trkC-IgG alone or along with different neurotrophinsat 10 ng/ml. Quantitation of viable VGNs was done in the same way as inFIG. 2 and the error bars are SEM.

[0019]FIG. 4 is a histogram depicting NT-4/5, BDNF and NT-3 protectionof VGNs from cisplatin neurotoxicity. VGNs were prepared from P5 ratsand maintained for 2 days in serum-free medium containing 1, 2 or 4μg/ml of cisplatin alone or together with 10 ng/ml of differentneurotrophins.

[0020] Quantitation of viable VGNs was done in the same way as in FIG.2. In some cultures in which high concentrations of the ototoxin wereadded, cell counts were performed from the entire area of the LabTekculture wells as overall number of viable neurons was low. In theseexperiments cell counts were performed in the same way for controlcultures. Data were collected from triplet cultures, and normalized as apercentage of the number of viable VGNs in the control cultures in eachof the experiments. The error bars stand for SEM. As compared to theculture containing cisplatin alone, NT-3, BDNF and NT-4/5 all showedvery significant protecting effects at all three doses (p<0.001). Whileneuroprotection by NT-4/5 and BDNF was statistically equivalent, thedifference between cultures containing NT-3 and cultures containingNT-4/5 (or BDNF) was highly significant (p<0.001). Abbreviation: Cis,Cisplatin.

[0021]FIG. 5 is a histogram depicting NT-4/5, BDNF and NT-3 protectionof VGNs against gentamicin neurotoxicity. VGNs were prepared from P5rats and maintained for 2 days in serum-free medium containing 1, 2 or 3mg/ml of gentamicin alone or combined with 10 ng/ml of differentneurotrophins. Quantitation of viable VGNs was done in the same way asin FIG. 4. Error bars are SEM. As compared to the culture containinggentamicin alone, NT-3, BDNF and NT-4/5 all showed very significantprotective effects at all three doses of gentamicin (p<0.001). Nosignificant difference was found between the protective effect of NT-3against gentamicin and that of NT-4/5 (or BDNF) except for the culturescontaining 1 mg/ml of gentamicin (p<0.05).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] In general, the following words or phrases have the indicateddefinition when used in the description, examples, and claims:

[0023] “Non-immunogenic in a human” means that upon contacting thepolypeptide in a pharmaceutically acceptable carrier and in atherapeutically effective amount with the appropriate tissue of a human,no state of sensitivity or resistance to the polypeptide is demonstrableupon the second administration of the polypeptide after an appropriatelatent period (e.g., 8 to 14 days).

[0024] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) neuron-damge-related balance impairment, preferablyototoxin-induced or inducible. Those in need of treatment include thosealready experiencing a balance impairment, those prone to having theimpairment, and those in which the impairments are to be prevented. Thebalance impairments are due to neuronal damage, preferably ototoxicity,wherein the damage is caused by infections, mechanical injury, loudsounds, aging, or chemical-induced ototoxicity, wherein in the case ofototoxins includes therapeutic drugs including antineoplastic agents,and aminoglycoside antibiotics, contaminants in foods or medicinals, andenvironmental or industrial pollutants. Typically, treatment isperformed to prevent or reduce ototoxicity, especially resulting from orexpected to result from administration of therapeutic drugs.

[0025] Preferably a therapeutically effective composition is givenimmediately after the exposure to prevent or reduce the ototoxic effect.More preferably, treatment is provided prophylactically, either byadministration of the composition prior to or concomitantly with theototoxic pharmaceutical or the exposure to the ototoxin.

[0026] By “ototoxic agent” in the context of the present invention ismeant a substance that through its chemical action injures, impairs, orinhibits the activity of a component of the nervous system related tobalance. The list of ototoxic agents that cause balance impairmentsincludes, but is not limited to, neoplastic agents such as vincristine,vinblastine, cisplatin, taxol, or dideoxy-compounds, e.g.,dideoxyinosine; alcohol; metals; industrial toxins involved inoccupational or environmental exposure; contaminants of food ormedicinals; or over-doses of vitamins or therapeutic drugs, e.g.,antibiotics such as penicillin or chloramphenicol, or megadoses ofvitamins A, D, or B6. Other toxic agents can cause ototoxicity-inducingbalance impairment can be characterized by methods as taught herein. By“exposure to an ototoxic agent” is meant that the ototoxic agent is madeavailable to, or comes into contact with, a mammal. Exposure to a toxicagent can occur by direct administration, e g., by ingestion oradministration of a food, medicinal, or therapeutic agent, e.g., achemotherapeutic agent, by accidental contamination, or by environmentalexposure, e.g., aerial or aqueous exposure.

[0027] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial anti-ototoxic effect for an extended period of time.

[0028] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, horses, cats, cows, etc.Preferably, the mammal herein is human.

[0029] A “patient” for the purposes of the present invention includesboth humans and other mammals. Thus the methods are applicable to bothhuman therapy and veterinary applications.

[0030] By “balance impairment” is meant a neurologic disorder,oto-neurological, in which the patient displays, complains of, or isdiagnosed to have known diagnostic symptoms of a balance disorder,including ataxic gait, preferably grossly ataxic, inability to stand onone leg, or inability to walk heel-to-toe, inability to tandem walk, anddizziness or vertigo that are neurologically related. During vertigo thepatient may experience a subjective impression of movement in space(subjective vertigo) or of objects moving in space (objective vertigo)usually with a loss of equilibrium. These impairments of interest to thepresent invention are those typically associated with damge to neurorns,and possibly hair cells, of the vestibular system related to the 8thcranial nerve. Particularly affected may be neurons of the vestibule,semicircular canal, 8th nerve, vestibular neurons of the brainstem andtheir temporal lobe connections, and more particularly the organ ofCorti.

[0031] Ototoxicity-related balance impairments include Meniere'ssyndrome, myringitis, otitis media, acute vestibular neuronitis, herpeszoster oticus, labyrinthitis, middle ear or labyrinthe tumors,petrositis, and otosclerosis. Incorporated herein by reference isChapters 196, 197, 198 and 199 of The Merck Index, 14th Edition, (1982),Merck Sharp & Dome Research Laboratories, N.J.) relating to descriptionand diagnosis of oto-neurological balance impairments.

[0032] Balance impairments include patients diagnosed with vestibularneuronitis. Tests are known and available for diagnosing balanceimpairments, such as vestibular problems, susceptible to the treatmentdisclosed herein. Neuro-otological, neuro-ophthalmological, neurologicalexaminations, and electro-oculography can be used. (Wennmo et al. ActaOtolaryngol (1982) 94:507-15).

[0033] Sensitive and specific measures are available to identifypatients with vestibular impairments. Dynamic and static platformposturography can be used for detecting vestibular disorders. Thesensory organization test (SOT) of dynamic posturography (EquiTest), themotor “perturbation” test, and Romberg's tests on a static (fixed) forceplatform each had over 90% specificity. The sensitivity of the SOT wasevaluated across five studies involving a total of 836 patients withperipheral vestibular deficits (PVDs). Abnormalities in the SOT weredetected in only 40% (n =338) of the cases. Static platformposturography sensitivity was evaluated across six studies involving atotal of 571 patients with PVDs, and abnormalities were detected in 53%(n =302) of these cases. Tests of spontaneous and positional nystagmusand the horizontal component of the vestibuloocular reflex (VOR), bycomparison, detected PVDs in 48% of 798 patients with suspectedvestibular impairment. For patients with vestibular deficits associatedwith central nervous system disease, a total of 389 cases wereidentified in five studies and SOT abnormalities were found in 54% (n=209) of these cases. The motor perturbation test was abnormal in 35% (n=41) of 119 patients with central vestibular disease. In conclusion, thesensitivity of static posturography appeared to be slightly better thanthat of dynamic posturography for detecting PVDs, but the level ofsensitivity for each posturography test, as well as for tests ofhorizontal VOR function, was considered to be low. Combining either typeof posturography with other tests of vestibular function, however,increased the overall sensitivity of detecting vestibular deficits to61% to 89%. It was concluded that dynamic and static platformposturography as well as tests of VOR function lack adequate sensitivityto detect vestibular impairment when applied in isolation. Posturographyappears to detect vestibular deficits in some patients who had normalVOR assessments and, therefore, provides supplemental rather thanredundant information about vestibular dysfunction. (Di Fabio, Phys.Ther.(1995) 75:290-305).

[0034] In one embodiment the invention constitutes a method for treatinga mammal having or prone to a balance impairment or treating a mammalprophylactically to prevent or reduce the occurrence or severity of abalance impairment that would result from exposure to an neuronalinjury, loss, or degeneration, preferably caused by an ototoxic agent,wherein a therapeutically effective amount of a trkB or trkC agonist isadministered to the mammal. Preferably the agonist is a neurotrophin,more preferably neurotrophin NT-4/5, NT-3, or BDNF, a functionalfragment, fusion or derivative thereof, such as a chimeric neurotrophin(having both trkB and trkC agonsim), a pantropic neurotrophin, or asmall molecule or antibody agonist thereof, as discussed in detailherein. Most preferably the agonist is NT-4/5 or a chimeric or pantropicvariant thereof having at least both trkB and trkC agonist activity. Apreferred chimeric or pantropic neurotrophin has a region conferringNT-3-receptor binding specificity and a region conferringNT-4/5-receptor binding specificity. A preferred pantropic neurotrophinis MNTS-1. In a preferred embodiment the binding of a chimeric orpantropic neurotrophin to a neurotrophic receptor is at least 80% of thebinding of the natural neurotrophin ligand to the receptor. When thepatient is human, the neurotrophins are preferably human neurotrophinsor derived from human neurotrophin sequences, in part to avoid orminimize recognition of the agonist as foreign. Optionally, the trkB ortrkC agonist is administered alone or in combination. Additionaloptional components include a hair cell growth factor or agonist, whichare compounds known to promote hair cell survival or prevent or reducecytotoxicity of hair cells. The method of the invention are particularlyeffective when the balance impairment is ototoxin induced or inducible.Preferably the neurons effected are vestibular ganglion neurons,preferably of Type I.

[0035] In one embodiment is a method for treating wherein theototoxicity results from administration of a therapeutically effectiveamount of an ototoxic pharmaceutical drug. Typical ototoxic drugs arechemotherapeutic agents, e.g. antineoplastic agents, and antibiotics.Other possible candidates include loop-diuretics, quinines or aquinine-like compound, and salicylate or salicylate-like compounds.

[0036] The methods of the invention are particularly effective when theototoxic compound is an antibiotic, preferably an aminoglycosideantibiotic. Ototoxic aminoglycoside antibiotics include but are notlimited to neomycin, paromomycin, ribostamycin, lividomycin, kanamycin,amikacin, tobramycin, viomycin, gentamicin, sisomicin, netilmicin,streptomycin, dibekacin, fortimicin, and dihydrostreptomycin, orcombinations thereof. Particular antibiotics include neomycin B,kanamycin A, kanamycin B, gentamicin Cl, gentamicin Cla, and gentamicinC2.

[0037] Balance impairments induced by aminoglycosides can be preventedor reduced by the methods of the invention. Although the aminoglycosidesare particularly useful due to their rapid bactericidal action ininfections by susceptible organisms, their use is limited to moresevere, complicated infections because of ototoxic and nephrotoxicside-effects. For this reason the aminoglycosides are considered to havea low therapeutic/risk ratio compared to other antibiotics usedsystemically. Aminoglycosides are a class of compounds characterized bythe ability to interfere with protein synthesis in micro-organisms.Aminoglycosides consist of two or more amino sugars joined in aglycoside linkage to a hexose (or aminocyclitol) nucleus. The hexosenuclei thus far known are either streptidine or 2-deoxystreptamine,though others may be anticipated. Aminoglycoside families aredistinguished by the amino sugar attached to the aminocyclitol. Forexample, the neomycin family comprises three amino sugars attached tothe central 2-deoxystreptamine. The kanamycin and glutamicin familieshave only two amino sugars attached to the aminocyclitol.Aminoglycosides include: neomycins (e.g. neomycin B and analogs andderivatives thereof), paromomycin, ribostamycin, lividomycin, kanamycins(e.g. kanamycin A, kanamycin B, and analogs and derivatives thereof),amikacin, tobramycin, viomycin, gentamicin (e.g., gentamicin C1,gentamicin C 1a, gentamicin C2, and analogs and derivatives thereof),sisomicin, netilmicin, streptomycin, dibekacin, fortimicin, anddihydrostreptomycin.

[0038] The aminoglycoside antibiotic which can be employed inconjunction with the ototoxicity inhibiting compositions of theinvention is any aminoglycoside antibiotic. Examples of suchaminoglycoside antibiotics include kanamycin (Merck Index 9th ed.#5132), gentamicin (Merck Index 9th ed. #4224), amikacin (Merck Index9th ed. #A1), dibekacin (Merck Index 9th ed. #2969), tobramycin (MerckIndex 9th ed. #9193), streptomycin (Merck Index 9th ed. #8611/8612),paromomycin (Merck Index 9th ed. #6844), sisomicin (Merck Index 9th ed.#8292), isepamicin and netilmicin, all known in the art. The usefulantibiotics include the several structural variants of the abovecompounds (e.g. kanamycin A, B and C; gentamicin A, Cl, C la, C2 and D;neomycin B and C and the like). The free bases, as well aspharmaceutically acceptable acid addition salts of these aminoglycosideantibiotics, can be employed.

[0039] For the purpose of this disclosure, the terms “pharmaceuticallyacceptable acid addition salt” shall mean a mono or poly salt formed bythe interaction of one molecule of the aminoglycoside antibiotic withone or more moles of a pharmaceutically acceptable acid. Included amongthose acids are acetic, hydrochloric, sulfuric, maleic, phosphoric,nitric, hydrobromic, ascorbic, malic and citric acid, and those otheracids commonly used to make salts of amine-containing pharmaceuticals.

[0040] Accordingly, the methods and compositions of the invention finduse for the prevention and treatment of opportunistic infections inanimals and man which are immunosuppressed as a result of eithercongenital or acquired immunodeficiency or as a side-effect ofchemotherapeutic treatment.

[0041] According to an alternate embodiment of the present invention, atrkB or trkC agonists is used advantageously in combination with a knownantimicrobial agent to provide improved methods and compositions toprevent and/or treat diseases induced by gram positive bacteriaincluding, but not limited to: Staphylococcus aureus, Streptococcuspneumonia, Hemophilus influenza; gram negative bacteria including, butnot limited to: Escherichia coli; Bacterium enteritis, Francisellatularensis; acid-fast bacteria including, but not limited toMycobacterium tuberculosis, and Mycobacterium leprae. Use of acombination of an antimicrobial agent together with a trkB or trkCagonist is advantageous with antibacterial aminoglycosides such asgentamicin, streptomycin, and the like which are known to have seriousototoxicity, which reduce the usefulness of such antimicrobial agents.Use of trkB or trkC agonist in combination with such agents permits alower dosage of the toxic antimicrobial agents while still achievingtherapeutic (antibacterial) effectiveness.

[0042] In some embodiments the trkB or trkC agonist is co-administeredwith an ototoxin. For example, an improved method is provided fortreatment of infection of a mammal by administration of anaminoglycoside antibiotic, the improvement comprising administering atherapeutically effective amount of a trkB or trkC agonist to thepatient in need of such treatment to reduce or prevent ototoxin-inducedbalance impairment associated with the antibiotic. In yet anotherembodiment is provided an improved method for treatment of cancer in amammal by administration of a chemotherapeutic compound, the improvementcomprises administering a therapeutically effective amount of a trkB ortrkC agonist to the patient in need of such treatment to reduce orprevent ototoxin-induced balance impairment associated with thechemotherapeutic drug.

[0043] Also provided herein are methods for promoting vestibularganglion neuron survival upon, prior to, or after exposure to an agentor effect that is capable of inducing a neuronal-injury-related balanceimpairment. Such agents and effects are those described herein. Themethod includes the step of administering to the neuron an effectiveamount of trkB or trkC agonist or other such compositions as discussedherein. Preferably, the method is used upon, prior to, or after exposureto a balance-impairing ototoxin.

[0044] In another preferred embodiment the ototoxic agent is achemotherapeutic agent, an antineoplastic agent. Preferred agentsinclude but are not limited to cisplatin or cisplatin-like compoundsBalance impairments resulting from the administration ofchemotherapeutic agents can be prevented or reduced by the methods ofthe invention. Ototoxic chemotherapeutic agents amenable to the methodsof the invention include, but are not limited to, cisplatin, taxol, andother chemotherapeutic agents believed to cause ototoxin-induced balanceimpairments, e.g., vincristine, an antineoplastic drug used to treathematological malignancies and sarcomas. The ototoxicity isdose-related.

[0045] Balance impairments resulting from administration of diureticscan be prevented or reduced by the methods of the invention. Diuretics,particularly “loop” diuretics, i.e. those that act primarily in the Loopof Henle, are candidate ototoxins. Illustrative examples, not limitingto the invention method, include furosemide, ethacrynic acid, andmercurial. Diuretics are typically used to prevent or eliminate edema.Diuretics are also used in nonedematous states such as hypertension,hypercalcemia, idiopathic hypercalciuria, and nephrogenic diabetesinsipidus.

[0046] In one embodiment the trkB or trkC agonist is administered priorto administration or exposure to a balance-impairing event such asexposure to an ototoxin.

[0047] In another embodiment the trkB or trkC agonist is administeredwith an agent that promotes hair cell growth or regeneration.

[0048] Preparation and Identification of Agonists

[0049] Agonists to trkB or trkC can be prepared by using the knownfamily of ligands for trkB or trkC. Survival of developing sensoryneurons is dependent upon trophic factors derived from their targettissues (Davies et al., 1986). Generally, a neurotrophin is a proteininvolved in the development, regulation and maintenance of the nervoussystem, and in particular of neurons. Currently, there are at least fiveknown important neurotrophic factors: nerve growth factor (NGF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4/5, also sometimes calledneurotrophin-5 (NT-5) or NT-4/5), brain-derived neurotrophic factor(BDNF), and ciliary neurotrophic factor (CNTF). The best characterizedmammalian neurotrophic factors are members of the nerve growth factor(NGF) family of proteins, and are called neurotrophins. These includeNGF (Levi-Montalcini, 1987), brain-derived neurotrophic factor (BDNF)(Barde et al., 1982; Leibrock et al., Nature (1989) 341:149)neurotrophin-3 (NT-3) (Hohn et al., Nature, 344: 339 (1990);Maisonpierre et al., Science, 247: 1446 (1990); Rosenthal et al.,Neuron, 4: 767 (1990); copending U.S. Ser. No. 07/494,024 filed Mar. 15,1990; U.S. application Ser. No. 07/490,004, filed Mar. 7, 1990; Ernforset al., 1990; Jones and Reichardt, 1990) and neurotrophin-4/5 (NT-4/5)(Berkemeier et al., 1991; Ip et al., 1992) and neurotrophin-6 (NT-6).

[0050] While NT-6 is newly cloned from Xenopus (Gotz et al., 1994) andis less well understood, it is now well accepted that the other fourmammalian neurotrophins exert their biological functions throughactivation of high-affinity binding receptors, the trks (Barbacid, 1993;Snider, 1994). Each of the neurotrophins binds to specific high-affinityreceptors, the trks (Klein et al., 1990; Kaplan et al., 1991; Klein etal., 1991a; Klein et al., 1991b; Soppet et al., 1991; Squinto et al.,1991; Lamballe et al., 1991; Tsoulfas et al., 1993; Ip et al., 1993).For example, NGF selectively binds to trkA, BDNF and NT-4/5 to trkB, andNT-3 to trkC. Although neurotrophins exert their main effects throughbinding to the trks, they also bind to the NGF low affinity receptor,P75. Recent studies indicate that the binding of NGF to P75 may enhancethe trkA-mediated signal transduction pathway (Davies et al., 1993a;Verdi et al., 1994; Barker and Shooter, 1994; Clary and Reichardt,1994).

[0051] Neurotrophins transduce intracellular signalling at least in partthrough the ligand-dependent activation of a class of tyrosinekinase-containing receptors of M =140-145,000 known as the trks(Martin-Zanca, et al. (1989); Kaplan, et al. (1991) Nature; Klein, etal.(1991a); Kaplan, et al. (1991) Science); Klein, et al. (1991b) Cell;Soppet, et al. (1991); Squinto, et al. (1991); Lamballe, et al. (1991);Tsoulfas, et al. (1993)). Thus, the signal transduction pathway ofneurotrophins is initiated by this high-affinity binding to andactivation of specific tyrosine kinase receptors and subsequent receptorautophosphorylation (Cordon-Cardo, et al. (1991)). Although there issome degree of cross-receptor interaction between the neurotrophins andthe different trks, the predominant specificity appears to be NGF/trkA,BDNF/trkB, and NT-3/trkC while NT-4/5 appears to interact primarily withtrkB as efficiently as BDNF (see above and Klein, et al. (1992); Klein,et al. (1989)).

[0052] Expression of trkB, trkC and p75 mRNAs in embryoniccochleovestibular ganglia (Ylikoski et al., 1993; Schecterson andBothwell, 1994) and BDNF and NT-3 mRNAs in the inner ear structures(Pirvola et al., 1992; Wheeler et al., 1994; Schecterson and Bothwell,1994) suggest a possible role of neurotrophins in the development ofVGNs and maintenance of VGNs in the adult. However, the expression ofneurotrophin receptors at the protein level has not been well determinedand the effects of the four neurotrophins have not been carefullycompared on VGNs. For example, the effects of NT-4/5 on the survival ofpostnatal VGNs have not been studied. In the present application,immunohistochemical evidence demonstrates that VGNs make trkB and p75,but not trkA proteins. Addition of NT-4/5, BDNF or NT-3 to the culturesenhanced postnatal rat VGN survival. In contrast, NGF showed nodetectable effects on survival of VGNs. The survival-promoting effectsof NT-4/5 (or BDNF) and NT-3 were specifically abolished by theirspecific antagonists trkB-IgG and trkC-IgG fusion proteins (Shelton etal., 1995), respectively. Furthermore, evidence presented hereindemonstrates that ototoxins including cisplatin and gentamicin inducedcell death of VGNs in normal cultures in a dose-dependent manner andthat NT-4/5, BDNF and NT-3, but not NGF, prevented or reduced theseneurotoxic effects.

[0053] In situ hybridization studies indicate that mRNAs for trkB andtrkC are expressed by embryonic cochleovestibular ganglia (Ylikoski etal., 1993; Schecterson and Bothwell, 1994) and that mRNAs for BDNF andNT-3 are found in the inner ear including organ of Corti (Pirvola etal., 1992; Wheeler et al., 1994; Schecterson and Bothwell, 1994).However, the expression patterns of neurotrophin receptors have not beenwell determined at the protein level and no study has compared thebiological effects of the four neurotrophins on VGNs. In particular, thesurvival-promoting effects of NT-4/5 have not been tested on VGNs.

[0054] DNA sequences encoding NGF, BDNF and NT-3 have all been isolated(ulIrich et al., Nature 303:821-825; Hyman et al., WO 91/03568; Hohn etal., WO 91/03569; and Kaisho et al., FEBS Letters 266:187-191).Researchers have transformed animal and non-animal hosts with thesesequences in order to express the neurotrophins.

[0055] Researchers have expressed human NGF, BDNF and NT-3 in mammalianexpression systems. Bruce and Heinrich (1989, Neurobiology of Aging10:89-94) expressed a DNA sequence encoding the complete precursor forhNGF in COS cells and detected hNGF dimer in the conditioned medium.However, they could not determine the efficiency at which pre-pro-hNGFwas converted to mature hNGF. Kakinuma et al (EP 0 414 151, 1991)expressed active hNGF in CHO cells. Hyman et al. (WO 91/03568, 1991)expressed hBDNF in CHO cells. Nakahama et al. (EP 0 386 752, 1990) andHohn et al. (WO 91/03569, 1991) expressed hNT-3 in COS cells.

[0056] U.S. Pat. Nos. 5,235,043 and 5,229,500 disclose human BDNFsequence and methods for its production and formulation. Applicant'sU.S. patent application Ser. No. ______ identified as Attorney DocketNumber P0980 and entitled “Treatment of Hearing Impairments” is alsoincorporated herein by reference.

[0057] NT-4/5, and its chimeric or pantropic neurotrophins, are mostpreferred agonists for use in the methods and compositions of thepresent invention. Its human gene and amino acid sequence are known(U.S. Pat. No. 5,364,769, which is incorporated herein by reference).NT-4/5 is defined to be a polypeptide encoded by the known mature humanNT-4/5 nucleotide sequence set forth in U.S. Pat. No. 5,364,769,fragments thereof having greater than about 5 residues comprising animmune epitope or other biologically active site of NT-4/5, amino acidsequence variants of said sequence, wherein an amino acid residue hasbeen inserted N- or C-terminal to, or within, said sequence or itsfragment as defined above, and/or amino acid sequence variants of saidsequence or its fragment as defined above wherein an amino acid residueof said sequence or fragment thereof has been substituted by anotherresidue, including other animal species of NT-4/5 such as ratpreproNT-4/5, and derivatives of NT-4/5 or its fragments as definedabove wherein the NT-4/5 or its fragments have been covalently modifiedby substitution with a moiety other than a naturally occurring aminoacid; provided, however, that such fragment or variant is novel andunobvious over the prior art, and is not NGF, BDNF, or NT-3 of anyanimal species or any known fragment of such NGF, BDNF, or NT-3. MatureNT-4/5 amino acid sequence variants generally will be about 75% (andusually >85%) homologous on an identical residue basis after aligning(introducing any necessary spaces) to provide maximum homology.

[0058] NT-4/5 nucleic acid is defined as RNA or DNA which encodes aNT-4/5 polypeptide or which hybridizes to such DNA and remains stablybound to it under stringent conditions and is greater than about 10bases in length; provided, however, that such hybridizing nucleic acidis novel and unobvious over any prior art nucleic acid including thatwhich encodes or is complementary to nucleic acid encoding BDNF, NT-3,or NGF. Stringent conditions are those which (1) employ low ionicstrength and high temperature for washing, for example, 0.15 MNaCl/0.015 M sodium citrate/0.1% NaDodSO₄ at 50° C., or (2) use duringwashing a denaturing agent such as formamide, for example, 50% (vol/vol)formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mMNaCl, 75 mM sodium citrate at 42° C.

[0059] DNA encoding NT-4/5 is obtained from brain tissue cDNA libraries,or genomic DNA, or by in vitro synthesis. Hybridizing nucleic acidgenerally is obtained by in vitro synthesis. Identification of NT-4/5DNA most conveniently is accomplished by probing human cDNA or genomiclibraries by labeled oligonucleotide sequences selected from the knownsequence in accord with known criteria, among which is that the sequenceshould be of sufficient length and sufficiently unambiguous that falsepositives are minimized. Typically, a ³²P-labeled oligonucleotide havingabout 30 to 50 bases is sufficient, particularly if the oligonucleotidecontains one or more codons for methionine or tryptophan. Isolatednucleic acid will be DNA that is identified and separated fromcontaminant nucleic acid encoding other polypeptides from the source ofnucleic acid. The nucleic acid may be labeled for diagnostic purposes.

[0060] Amino acid sequence variants of NT-4/5 are prepared byintroducing appropriate nucleotide changes into the NT-4/5 DNA, or by invitro synthesis of the desired NT-4/5. Such variants include, forexample, deletions from, or insertions or substitutions of, residueswithin the amino acid sequence for human NT-4/5. Any combination ofdeletion, insertion, and substitution is made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics.

[0061] The amino acid changes also may result in further modificationsof NT-4/5 upon expression in recombinant hosts, e.g. introducing ormoving sites of glycosylation, or introducing membrane anchor sequences(in accordance with U.S. Ser. No. 07/083,757, filed Aug. 6, 1987, whichis equivalent to PCT WO 89101041 published Feb. 9, 1989).

[0062] There are two principal variables in the construction of aminoacid sequence variants: the location of the mutation site and the natureof the mutation. These are variants may represent naturally occurringalleles (which will not require manipulation of the NT-4/5 DNA) orpredetermined mutant forms which are made by mutating the DNA, either toarrive at an allele or a variant that is not found in nature. Ingeneral, the location and nature of the mutation chosen will depend uponthe NT-4/5 characteristic to be modified. For example, candidate NT-4/5antagonists or super agonists will be initially selected by locatingsites that are identical or highly conserved among NGF, BDNF, NT-3, andNT-4/5. These sites then will be modified in series, e.g., by (1)substituting first with conservative choices and then with more radicalselections depending upon the results achieved, (2) deleting the targetresidue, or (3) inserting residues of the same or different classadjacent to the located site, or combinations of options 1-3.

[0063] One helpful technique is called “ala scanning”. Here, a residueor group of target residues 3.0 are identified and substituted byalanine or polyalanine. Those domains demonstrating functionalsensitivity to the alanine substitutions then are refined by introducingfurther or other variants at or for the sites of alanine substitution.Obviously, such variations which, for example, convert NT-4/5 into NGF,BDNF, or NT-3 are not included within the scope of this invention, norare any other NT-4/5 variants or polypeptide sequences that are notnovel and unobvious over the prior art. Thus, while the site forintroducing an amino acid sequence variation is predetermined, thenature of the mutation per se need not be predetermined. For example, tooptimize the performance of a mutation at a given site, ala scanning orrandom mutagenesis is conducted at the target codon or region and theexpressed NT-4/5 variants are screened for the optimal combination ofdesired activity.

[0064] Amino acid sequence deletions generally range from about 1 to 30residues, more preferably about 1 to 10 residues, and typically arecontiguous. Deletions may be introduced into regions of low homologyamong BDNF, NGF, NT-3, and NT-4/5 to modify the activity of NT-4/5.Deletions from NT-4/5 in areas of substantial homology with BDNF, NT-3,and NGF will be more likely to modify the biological activity of NT-4/5more significantly. The number of consecutive deletions will be selectedso as to preserve the tertiary structure of NT-4/5 in the affecteddomain, e.g., beta-pleated sheet or alpha helix.

[0065] Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a thousand or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Intrasequence insertions (i.e., insertions within the mature NT-4/5sequence) may range generally from about 1 to 10 residues, morepreferably 1 to 5, most preferably 1 to 3. An example of a terminalinsertion includes fusion of a heterologous N-terminal signal sequenceto the N-terminus of the NT-4/5 molecule to facilitate the secretion ofmature NT-4/5 from recombinant hosts. Such signals generally will behomologous to the intended host cell and include STII or lpp for E.coli, alpha factor for yeast, and viral signals such as herpes gD formammalian cells. Other insertions include the fusion of an immunogenicpolypeptide such as a bacterial or yeast protein to the N- or C-terminiof NT-4/5.

[0066] The third group of variants are those in which at least one aminoacid residue in the NT-4/5 molecule, and preferably only one, has beenremoved and a different residue inserted in its place. In someembodiments substitutions of one to five amino acids are made. In yetanother embodiment one to three amino acids are substituted. In somepreferred embodiments two amino acid substitutions are made. Thesubstitutions can be chosen from the table herein. An example is thereplacement of arginine and lysine by other amino acids to render theNT-4/5 resistant to proteolysis by serine proteases, thereby creating amore stable NT-4/5 analogue. The sites of greatest interest forsubstitutional mutagenesis include sites where the amino acids found inBDNF, NGF, NT-3, and NT-4/5 are substantially different in terms of sidechain bulk, charge or hydrophobicity, but where there also is a highdegree of homology at the selected site within various animal analoguesof NGF, NT-3, and BDNF (e.g., among all the animal NGFs, all the animalNT-3s, and all the BDNFs). This analysis will highlight residues thatmay be involved in the differentiation of activity of the trophicfactors, and therefore, variants at these sites may affect suchactivities. Examples of such NT-4/5 sites, numbered from the matureN-terminal end, and exemplary substitutions include NT-4/5 (G₇₈→K, H, Qor R) and NT-4/5 (R₈₅→E, F, P, Y or W). Other sites of interest arethose in which the residues are identical among all animal species'BDNF, NGF, NT-3, and NT-4/5, this degree of conformation suggestingimportance in achieving biological activity common to all four factors.These sites, especially those falling within a sequence of at least 3other identically conserved sites, are substituted in a relativelyconservative manner. Such conservative substitutions are shown in Table1 under the heading of preferred substitutions. If such substitutionsresult in a change in biological activity, then more substantialchanges, denominated exemplary substitutions in Table 1, or as furtherdescribed below in reference to amino acid classes, are introduced andthe products screened. TABLE 1 Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) val; leu; ile val           Arg (R)     lys; gln; asn           lys Asn (N)      gln; his; lys; arg          gln Asp (D)      glu           glu Cys (C)      ser          ser Gln (Q)      asn           asn Glu (E)      asp          asp Gly (G)      pro           pro His (H)      asn; gln; lys;arg;           arg Ile (I)      leu; val; met; ala; phe;      norleucineleu           Leu (L) norleucine; ile; val;     met; ala; phe          ile Lys (K)      arg; gln; asn           arg Met (M)      leu;phe; ile           leu Phe (F)      leu; val; ile; ala           leu Pro(P)      gly           gly Ser (S)      thr           thr Thr (T)     ser           ser Trp (W)      tyr           tyr Tyr (Y)      trp;phe; thr; ser           phe Val (V)      ile; leu; met; phe;     ala;norleucine leu          

[0067] Sites particularly suited for conservative substitutions include,numbered from the N-terminus of the mature NT-4/5, R11, G12, E13, V16,D18, W23, V24, D26, V40, L41, Q54, Y55, F56, E58, T59, G77, R79, G80,H85, W86, A99, L100, T101, W110, R111, W112, I113, R114, I115, D116, andT118. Cysteine residues not involved in maintaining the properconformation of NT-4/5 also may be substituted, generally with serine,in order to improve the oxidative stability of the molecule and preventaberrant crosslinking. Sites other than those set forth in thisparagraph are suitable for deletional or insertional studies generallydescribed above.

[0068] Substantial modifications in function or immunological identityare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Naturallyoccurring residues are divided into groups based on common side chainproperties:

[0069] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0070] (2) neutral hydrophilic: cys, ser, thr;

[0071] (3) acidic: asp, glu;

[0072] (4) basic: asn, gln, his, lys, arg;

[0073] (5) residues that influence chain orientation: gly, pro; and

[0074] (6) aromatic: trp, tyr, phe.

[0075] Non-conservative substitutions will entail exchanging a member ofone of these classes for another. Such substituted residues also may beintroduced into the conservative substitution sites set forth above or,more preferably, into the remaining (non-conserved) sites.

[0076] Examples of NT-4/5 variants include NT-4/5(65NAE67→NAS or NAT)(this adds an N-linked glycosylation site); NT-4/5(R83-Q94);NT-4/5(G1-C61) (variants so depicted are fragments containing theresidues indicated); NT-4/5(G1-C17); NT-4/5(C17-C61); NT-4/5(C17-C78);NT-4/5(C17-C90); NT-4/5(C17-C119); NT-4/5(C17-C121); NT-4/5(R11-R27);NT-4/5(R11-R34); NT-4/5(R34-R53); NT-4/5(C61-C78); NT-4/5(R53-C61);NT-4/5(C61-C119); NT-4/5(C61-C78); NT-4/5(C78-C119); NT-4/5(C61-C90);NT-4/5(R60-C78); NT-4/5(K62-C119); NT-4/5(K62-K91); NT-4/5(R79-R98);NT-4/5(R83-K93); NT-4/5(T101-R111); NT-4/5(G1-C121) V L T V K R V R R;NT-4/5(V40-C121) V L T V K R V R R; NT-4/5(V40-C121)SLTIKRIRA;NT-4/5(V40-C121)TLSRKAGRRA; DDDSPIARRGEISVCDSVSDWVSAPDKDTAVDIKGDDVMVLKKVGINHSV; NT-4/5(V40-

[0077] C121); hNGF(S1-V48) NT-4/5(V40-C121) hNGF(V109-A120);BDNF(R7-Q48) NT-4/5(V40-C121) BDNF(V110-R119); NT-4/5(ΔC78);NT-4/5(ΔC61); NT-4/5(ΔQ54-ΔT59) (variants depicted in this fashioncomprise deletions of the indicated span of residues, inclusive);NT-4/5(ΔR60-ΔD82); NT-4/5(ΔH85-ΔS88); NT-4/5(ΔW86-ΔT101); NT-4/5(R53→H);NT-4/5(K91→H); NT-4/5(V108→F); NT-4/5(R84→Q, H, N, T, Y or W); andNT-4/5(D116→E, N, Q, Y, S or T).

[0078] Also included is NT-4/5 wherein position 70 is substituted withan amino acid residue other than G, E, D or P; position 71 with otherthan A, P or M; and/or position 83 with other than R, D, S or K; as wellas cyclized NT-4/5 fragments, including cyclic polypeptides comprisingthe sequences IKTG, EIKTG, EIKTGN, SPV, SPVK, HQV, KSS, KSSA, YAEHKS,RYAEHKS, RYAEHKSH, YAHKSH, ANRTS, NRT, ANRT, NRTS, KEA, KEAR, KEARP,IDDK, SENN, TSENN, TSENNK or KLVG.

[0079] Also within the scope hereof are BDNF, NT-3, and NGF amino acidvariants having analogous structures to the NT-4/5 variants set forthherein. For example, the analogous positions of NGF, NT-3, and BDNF aresubstituted with a residue other than D, E, or P, respectively, inanalogy to the same mutation at position 70 of NT-4/5.

[0080] DNA encoding NT-4/5 variants preferably is prepared bysite-specific mutagenesis of DNA that encodes an earlier preparedvariant or a nonvariant version of NT-4/5. Site-specific mutagenesisallows the production of NT-4/5 variants through the use of specificoligonucleotide sequences that encode the DNA sequence of the desiredmutation, as well as a sufficient number of adjacent nucleotides, toprovide a primer sequence of sufficient size and sequence complexity toform a stable duplex on both sides of the deletion junction beingtraversed. Typically, a primer of about 20 to 25 nucleotides in lengthis preferred, with about 5 to 10 residues on both sides of the junctionof the sequence being altered. In general, the technique ofsite-specific mutagenesis is well known in the art, as exemplified bypublications such as Adelman et al., DNA, 2: 183 (1983).

[0081] As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily commercially available and their use is generally well known tothose skilled in the art. Also, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol., 153: 3 [1987]) may be employed to obtain single-stranded DNA.Alternatively, nucleotide substitutions are introduced by synthesizingthe appropriate DNA fragment in vitro and amplifying it by polymerasechain reaction (PCR) procedures known per se in the art.

[0082] In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevant protein. Anoligonucleotide primer bearing the desired mutated sequence is prepared,generally synthetically, for example, by the method of Crea et al.,Proc. Natl. Acad. Sci. (USA), 75: 5765 (1978). This primer is thenannealed with the single-stranded protein-sequence-containing vector,and subjected to DNA-polymerizing enzymes such as E. coli polymerase IKlenow fragment, to complete the synthesis of the mutation-bearingstrand. Thus, a heteroduplex is formed wherein one strand encodes theoriginal non-mutated sequence and the second strand bears the desiredmutation. This heteroduplex vector is then used to transform appropriatecells such as JM101 cells and clones are selected that includerecombinant vectors bearing the mutated sequence arrangement.

[0083] After such a clone is selected, the mutated region may be removedand placed in an appropriate vector for protein production, generally anexpression vector of the type that is typically employed fortransformation of an appropriate host.

[0084] Most deletions and insertions, and substitutions in particular,are not expected to produce radical changes in the characteristics ofthe NT-4/5 molecule, and single substitutions will preserve at least oneimmune epitope in the NT-4/5 polypeptide.

[0085] Since it is often difficult to predict in advance thecharacteristics of a variant NT-4/5, it will be appreciated that somescreening will be needed to select the optimal variant. One can screenfor enhanced trophic activity, differential neuron cell typespecificity, stability in recombinant cell culture or in plasma (e.g.against proteolytic cleavage), possession of antagonist activity,oxidative stability, ability to be secreted in elevated yields, and thelike. For example, a change in the immunological character of the NT-4/5molecule, such as affinity for a given antibody, is measured by acompetitive-type immunoassay. Changes in the enhancement or suppressionof neurotrophic activities by the candidate mutants are measured bydendrite outgrowth or explant cell survival assays. Modifications ofsuch protein properties as redox or thermal stability, hydrophobicity,susceptibility to proteolytic degradation, or the tendency to aggregatewith carriers or into multimers are assayed by methods well known in theart.

[0086] Trypsin or other protease cleavage sites are identified byinspection of the encoded amino acid sequence for paired basic residues,e.g. combinations of adjacent arginyl and lysinyl residues. These arerendered inactive to protease by substituting one of the residues withanother residue, preferably a basic residue such as glutamine or ahydrophobic residue such as serine; by deleting one or both of the basicresidues; by inserting a prolyl residue immediately after the last basicresidue; or by inserting another residue between the two basic residues.

[0087] A variant NT-4/5 typically is made by site-specific mutagenesisof the native NT-4/5-encoding nucleic acid, expression of the variantnucleic acid in recombinant cell culture, and, optionally, purificationfrom the cell culture, for example, by bioassay of the variant'sactivity or by immunoaffinity adsorption on a rabbit polyclonalanti-NT-4/5 column (to absorb the variant by binding it to at least oneremaining immune epitope). Small fragments, on the order of 40 residuesor less, are conveniently made by in vitro methods.

[0088] The NT-4/5-encoding nucleic acid, whether variant or cDNA, thenis ligated into a replicable vector for further cloning or forexpression. Vectors are useful for performing two functions incollaboration with compatible host cells (a host-vector system). Onefunction is to facilitate the cloning of the nucleic acid that encodesthe NT-4/5, i.e., to produce usable quantities of the nucleic acid. Theother function is to direct the expression of NT-4/5. One or both ofthese functions are performed by the vector-host system. The vectorswill contain different components depending upon the function they areto perform as well as the host cell that is selected for cloning orexpression.

[0089] Each vector will contain nucleic acid that encodes NT-4/5 asdescribed above. Typically, this will be DNA that encodes the NT-4/5 inits mature form linked at its amino terminus to a secretion signal. Thissecretion signal preferably is the NT-4/5 presequence that normallydirects the secretion of NT-4/5 from human cells in vivo. However,suitable secretion signals also include signals from other animalNT-4/5, signals from NGF, NT-2, or NT-3, viral signals, or signals fromsecreted polypeptides of the same or related species.

[0090] If the signal sequence is from another NT molecule, it may be theprecursor sequence spanning from the initiating methionine (M) residueof NT-2, NT-3, or NGF up to the arginine (R) residue just before thefirst amino acid of the mature protein, or a consensus or combinationsequence from any two or more of those precursors taking into accounthomologous regions of the precursors. The DNA for such precursor regionis ligated in reading frame to DNA encoding the mature NT-4/5.

[0091] Expression and cloning vectors contain a nucleic acid sequencethat enables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomes, and includesorigins of replication or autonomously replicating sequences. Suchsequences are well-known for a variety of bacteria, yeast and viruses.The origin of replication from the well-known plasmid pBR322 is suitablefor most gram negative bacteria, the 2 μplasmid origin for yeast andvarious viral origins (SV40, polyoma, adenovirus, VSV or BPV) are usefulfor cloning vectors in mammalian cells. Origins are not needed formammalian expression vectors (the SV40 origin may typically be used onlybecause it contains the early promoter). Most expression vectors are“shuttle” vectors, i.e. they are capable of replication in at least oneclass of organisms but can be transfected into another organism forexpression. For example, a vector is cloned in E. coli and then the samevector is transfected into yeast or mammalian cells for expression eventhough it is not capable of replicating independently of the host cellchromosome.

[0092] DNA also is cloned by insertion into the host genome. This isreadily accomplished with bacillus species, for example, by including inthe vector a DNA sequence that is complementary to a sequence found inbacillus genomic DNA. Transfection of bacillus with this vector resultsin homologous recombination with the genome and insertion of NT-4/5 DNA.However, the recovery of genomic DNA encoding NT-4/5 is more complexthan that of an exogenously replicated vector because restriction enzymedigestion is required to excise the NT-4/5 DNA.

[0093] Expression and cloning vectors should contain a selection gene,also termed a selectable marker. This is a gene that encodes a proteinnecessary for the survival or growth of a host cell transformed with thevector. The presence of this gene ensures that any host cell whichdeletes the vector will not obtain an advantage in growth orreproduction over transformed hosts. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins, e.g.ampicillin, neomycin, methotrexate or tetracycline, (b) complementauxotrophic deficiencies, or (c) supply critical nutrients not availablefrom complex media, e.g. the gene encoding, D-alanine racemase forbacilli.

[0094] A suitable selection gene for use in yeast is the trp1 genepresent in the yeast plasmid YRp7 5 (Stinchcomb et al., 1979, Nature282:39; Kingsman et al., 1979, Gene 7:141; or Tschemper et al., 1980,Gene 10:157). The trp1 gene provides a selection marker for a mutantstrain of yeast lacking the ability to grow in tryptophan, for example,ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics 85:12). The presence ofthe trp1 lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2 10 deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

[0095] Examples of suitable selectable markers for mammalian cells aredihydrofolate reductase (DHFR) or thymidine kinase. Such markers enablethe identification of cells which were competent to take up the NT-4/5nucleic acid. The mammalian cell transformants are placed underselection pressure which only the transformants are uniquely adapted tosurvive by virtue of having taken up the marker. Selection pressure isimposed by culturing the transformants under conditions in which theconcentration of selection agent in the medium is successively changed,thereby leading to amplification of both the selection gene and the DNAthat encodes NT-4/5. Amplification is the process by which genes ingreater demand for the production of a protein critical for growth arereiterated in tandem within the chromosomes of successive generations ofrecombinant cells.

[0096] Increased quantities of NT-4/5 are synthesized from the amplifiedDNA.

[0097] For example, cells transformed with the DHFR selection gene arefirst identified by culturing all of the transformants in a culturemedium which lacks hypoxanthine, glycine, and thymidine. An appropriatehost cell in this case is the Chinese hamster ovary (CHO) cell linedeficient in DHFR activity, prepared and propagated as described byUrlaub and Chasin, 1980, Proc. Nat'l. Acad. Sci. USA 77:4216. Aparticularly useful DHFR is a mutant DHFR that is highly resistant toMTX (EP 117,060A). This selection agent can be used with any otherwisesuitable host, e.g. ATCC No. CCL61 CHO-K1, notwithstanding the presenceof endogenous DHFR. The DHFR and NT-4/5-encoding DNA then is amplifiedby exposure to an agent (methotrexate, or MTX) that inactivates theDHFR. One ensures that the cell requires more DHFR (and consequentlyamplifies all exogenous DNA) by selecting only for cells that can growin successive rounds of ever-greater MTX concentration.

[0098] Alternatively, hosts co-transformed with genes encoding NT-4/5,wild-type DHFR, and another selectable marker such as the neo gene canbe identified Using a selection agent for the selectable marker such asG418 and then selected and amplified using methotrexate in a wild-typehost that contains endogenous DHFR.

[0099] Other methods, vectors and host cells suitable for adaptation tothe synthesis of NT-4/5 in recombinant vertebrate cell culture aredescribed in M. J. Gething et al., Nature 293:620-625 (1981); N Manteiet al., Nature 281:40-46 (1979); and A. Levinson et al., EP 117,060A and117,058A. A particularly useful plasmid for mammalian cell cultureexpression of NT-4/5 is pRK5 (EP pub. no. 307,247) or pSVI6B (U.S. Ser.No. 07/441,574 filed Nov. 22, 1989).

[0100] Expression vectors, unlike cloning vectors, should contain apromoter which is recognized by the host organism and is operably linkedto the NT-4/5 nucleic acid. Promoters are untranslated sequences locatedupstream from the start codon of a structural gene (generally withinabout 100 to 1000 bp) that control the transcription and translation ofnucleic acid under their control. They typically fall into two classes,inducible and constitutive. Inducible promoters are promoters thatinitiate increased levels of transcription from DNA under their controlin response to some change in culture conditions, e.g. the presence orabsence of a nutrient or a change in temperature. At this time a largenumber of promoters recognized by a variety of potential host cells arewell known. These promoters are operably linked to NT-4/5-encoding DNAby removing them from their gene of origin by restriction enzymedigestion, followed by insertion 5′ to the start codon for NT-4/5. Thisis not to say that the genomic NT-4/5 promoter is not usable. However,heterologous promoters generally will result in greater transcriptionand higher yields of expressed NT-4/5.

[0101] Nucleic acid is operably linked when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein which participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,operably linked means that the DNA sequences being linked are contiguousand, in the case of a secretory leader, contiguous and in reading phase.Linking is accomplished by ligation at convenient restriction sites. Ifsuch sites do not exist then synthetic oligonucleotide adaptors orlinkers are used in accord with conventional practice.

[0102] Promoters suitable for use with prokaryotic hosts include theβ-lactamase and lactose promoter systems (Chang et al., 1978, Nature275:615; and Goeddel et al., 1979, Nature 281:544), alkalinephosphatase, a tryptophan (trp) promoter system (Goeddel, 1980, NucleicAcids Res. 8:4057 and EPO Appln. Publ. No. 36,776) and hybrid promoterssuch as the tac promoter (H. de Boer et al., 1983, Proc. Nat'l. Acad.Sci. USA 80:21-25). However, other known bacterial promoters aresuitable.

[0103] Their nucleotide sequences have been published, thereby enablinga skilled worker operably to ligate them to DNA encoding NT-4/5(Siebenlist et al. 1980, Cell 20:269) using linkers or adaptors tosupply any required restriction sites. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding NT-4/5.

[0104] Suitable promoting sequences for use with yeast hosts include thepromoters for 3-phosphoglycerate kinase (Hitzeman et al., 1980, J. Biol.Chem. 255:2073) or other glycolytic enzymes (Hess et al., 1968, J. Adv.Enzyme Reg. 7:149; and Holland, 1978, Biochemistry 17:4900), such asenolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase.

[0105] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin R. Hitzeman et al., EP 73,657A. Yeast enhancers also areadvantageously used with yeast promoters.

[0106] NT-4/5 transcription from vectors in mammalian host cells iscontrolled by promoters obtained from the genomes of viruses such aspolyoma, cytomegalovirus, adenovirus, retroviruses, hepatitis-B virusand most preferably Simian Virus 40 (SV40), or from heterologousmammalian promoters, e.g. the actin promoter. The early and latepromoters of the SV40 virus are conveniently obtained as an SV40restriction fragment which also contains the SV40 viral origin ofreplication (Fiers et al., 1978, Nature 273:113). Of course, promotersfrom the host cell or related species also are useful herein.

[0107] Transcription of NT-4/5-encoding DNA by higher eukaryotes isincreased by inserting an enhancer sequence into the vector. An enhanceris a nucleotide sequence, usually about from 10-300 bp, that acts on apromoter to increase its transcription and does so in a manner that isrelatively orientation and position independent. Many enhancer sequencesare now known from mammalian genes (globin, elastase, albumin,α-fetoprotein and insulin). Typically, however, one will use an enhancerfrom a eukaryotic cell virus. Examples include the SV40 enhancer on thelate side of the replication origin (bp 100-270), the cytomegalovirusearly promoter enhancer, the polyoma enhancer on the late side of thereplication origin, and adenoviral enhancers. The enhancer may bespliced into the vector at a position 5′ or 3′ to the NT-4/5-encodingsequence, but is preferably located at a site 5′ from the promoter.

[0108] Expression vectors used in eukaryotic host cells (yeast, fungi,insect, plant, animal, human or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′ untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain regions thatare transcribed as polyadenylated segments in the untranslated portionof the mRNA encoding NT-4/5. The 3′ untranslated regions also includetranscription termination sites.

[0109] Suitable host cells for cloning or expressing the vectors hereinare the prokaryote, yeast or higher eukaryote cells described above.Suitable prokaryotes include gram negative or gram positive organisms,for example E. coli or bacilli. A preferred cloning host is E. coli 294(ATCC 31,446) although other gram negative or gram positive prokaryotessuch as E. coli B, E. coli X1776 (ATCC 31,537), E. coli W3110 (ATCC27,325), pseudomonas species, or Serratia marcesans are suitable.

[0110] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable hosts for NT-4/5-encodingvectors. Saccharomyces cerevisiae, or common baker's yeast, is the mostcommonly used among lower eukaryotic host microorganisms. However, anumber of other genera, species and strains are commonly available anduseful herein.

[0111] Suitable host cells for the expression of NT-4/5 are derived frommulticellular organisms. Such host cells are capable of complexprocessing and glycosylation activities. In principle, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture, although cells from mammals such as humans arepreferred. Propagation of such cells in culture is per se well known.See issue Culture, Academic Press, Kruse and Patterson, editors (1973).Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary cell lines, the W138, BHK, COS-7, MDCK cell linesand human embryonic kidney cell line 293.

[0112] Host cells are transformed with the above-described expression orcloning vectors and cultured in conventional nutrient media modified asis appropriate for inducing promoters or selecting transformantscontaining amplified genes. The culture conditions, such as temperature,pH and the like, suitably are those previously used with the host cellselected for cloning or expression, as the case may be, and will beapparent to the ordinary artisan.

[0113] Covalent modifications of NT-4/5 molecules are included withinthe scope of this invention. Variant NT-4/5 fragments having up to about40 residues may be conveniently prepared by in vitro synthesis. Inaddition, covalent modifications are introduced into the molecule byreacting targeted amino acid residues of the NT-4/5 polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

[0114] Cysteinyl residues most commonly are reacted with α-haloacetates(and corresponding amines), such as chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid,chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide,methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0115] Histidyl residues are derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1M sodium cacodylateat pH 6.0.

[0116] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0117] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0118] The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using ¹²⁵I or ¹³¹I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

[0119] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

[0120] Derivatization with bifunctional agents is useful forcrosslinking NT-4/5 to a water-insoluble support matrix or surface foruse in the method for purifying anti-NT-4/5 antibodies, and vice versa.Commonly used crosslinking agents include, e g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidyl-propionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

[0121] Glutaminyl and asparaginyl residues are frequently deamidated tothe corresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

[0122] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co, San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group. NT-4/5 also is covalently linked to nonproteinaceouspolymers, e.g. polyethylene glycol, polypropylene glycol orpolyoxyalkylenes, in the manner set forth in U.S. Ser. No. 07/275,296 orU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

[0123] NT-4/5 preferably is recovered from the culture medium as asecreted protein, although it also may be recovered from host celllysates when directly expressed without a secretory signal. When NT-4/5is expressed in a recombinant cell other than one of human origin, theNT-4/5 is thus completely free of proteins of human origin. However, itis necessary to purify NT-4/5 from recombinant cell proteins in order toobtain preparations that are substantially homogeneous as to protein. Asa first step, the culture medium or lysate is centrifuged to removeparticulate cell debris. NT-4/5 thereafter is purified from contaminantsoluble proteins, for example, by fractionation on immunoaffinity or ionexchange columns; ethanol precipitation; reverse phase HPLC;chromatography on silica or on a cation exchange resin such as DEAE;chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; or gelelectrophoresis using, for example, Sephadex G-75. NT-4/5 variants inwhich residues have been deleted, inserted or substituted are recoveredin the same fashion as native NT-4/5, taking account of any substantialchanges in properties occasioned by the variation. For example,preparation of an NT-4/5 fusion with another protein, e.g. a bacterialor viral antigen, facilitates purification because an immunoaffinitycolumn containing antibody to the antigen can be used to adsorb thefusion. A protease inhibitor such as phenyl methyl sulfonyl fluoride(PMSF) also may be useful to inhibit proteolytic degradation duringpurification, and antibiotics may be included to prevent the growth ofadventitious contaminants. One skilled in the art will appreciate thatpurification methods suitable for native NT-4/5 may require modificationto account for changes in the character of NT-4/5 or its variants uponexpression in recombinant cell culture.

[0124] The trkB and trkC receptor DNA sequences are known. The receptorscan be expressed to obtain a soluble form of the receptor by identifyingthe extracellular domain and excising the transmembrane domaintherefrom). The soluble form of the receptor can then be used to screenfor trkB or trkC binding molecules, preferably small organic molecules,that are candidate agonists for receptor activity. Screening of agonistsuses, for example, transformed cells expressing trkB or trkC receptor.Further or alternative screening uses the assays taught herein.

[0125] As discussed above variants of native neurotrophins are made thatact as agonists. The receptor binding site(s) of a neurotrophin aredetermined by binding studies. These regions can be subcloned and testedfor agonist activity. Such regions can be also be constructed intolarger molecules using known protein engineering techniques, such astemplate-assembly synthesis.

[0126] Standard mutagenesis techniques (deletion or radical substitutionof appropriate nucleic acids) are used to identify such regions and tocreate mutants for testing for agonism. Agonist activity can bedetermined by several means, including the assays described herein.

[0127] Chimeric or pantropic neurotrophins that bind either trkB or trkCor preferably both are suitable for use in the methods and compositionsof the invention. By the term “pantropic neurotrophins” or “pantropicneurotrophic factors”, or grammatical equivalents, herein is meant aneurotrophin which, unlike naturally occurring neurotrophins, hasmultiple neurotrophin specificities. That is, it contains domains whichconfer different neurotrophin specificities. WO 95/33829 andcorresponding U.S. Ser. No. 08/253,937, are hereby incorporated byreference for describing, making and using pantropic neurotrophicfactors suitable for practicing the present invention. The discussionsherein pertaining to NT-4/5 or pantropic neurotrophin synthesis, design,expression and use apply to chimeric and other neurotrophins as well. Inone embodiment, this means that the pantropic neurotrophins of thepresent invention will bind to a variety of neurotrophic receptors.

[0128] Thus, for example, naturally occurring NGF, which is the naturalor native ligand for the trkA receptor, does not bind appreciably toeither the trkB or trkC receptor with high affinity; for example, NGFbinds to these receptors with a 500-1000 fold lower K_(D) than BDNF orNT-3, respectively. However, a pantropic NGF, i.e. a pantropicneurotrophin whose amino acid backbone is based on NGF, may bind to atleast the trkA, trkB and p75 receptor. Alternatively, a pantropic NGFwill bind to the trkA, trkC and p75 receptor. One preferred embodimentallows the binding of the trkA, trkB, trkC and p75 receptor. Similarly,naturally occurring BDNF and NT-4/5, which are the natural ligands forthe trkB receptor, do not bind appreciably to either the trkA or trkCreceptor as above. Thus pantropic BDNF or NT-4/5 will bind to trkB andany combination of trkA, trkC and p75, as shown above for pantropic NGF.

[0129] In alternative embodiments, the naturally occurring neurotrophinwill bind with poor affinity to several neurotrophin receptors. In thisembodiment, the pantropic neurotrophin binds to these receptors withaffinities higher than normally found, similar to the affinities seenfor the natural ligand. For example, NT-3 binds strongly to trkC, andweakly to trkA and trkB. Thus, a pantropic NT-3 binds to trkC with itsnormal binding affinity, and will bind to either trkA with an affinitysimilar to the trkA natural ligand, NGF, or to trkB with an affinitysimilar to the trkB natural ligands BDNF or NT-4/5, or both.

[0130] In a preferred embodiment, methods of treatment use a chimeric orpantropic neurotrophin or variant with a binding affinity forneurotrophin receptors at least about 50-60%, preferably about 75-80%,and even more preferably about 90%, and most preferably 100% of thebinding affinity of the natural ligand. Thus, a pantropic NGF will bindto the trkB or trkC receptor with at least 50% of the binding of BDNF orNT-4/5, or NT-3, respectively. This affinity is measured by a variety ofways, as will appreciated by those skilled in the art. The preferredmethod is the use of competition assays, as shown in (Hulme, et al.) andin Example 2. Generally, binding affinities are reported as IC₅₀, thatis, the concentration of unlabeled competitor which inhibits 50% of thebinding of labeled ligand to the receptor.

[0131] In alternative embodiments, the pantropicity of the neurotrophinis measured not by binding affinity to neurotrophin receptors, butrather by the neuronal survival or neurite outgrowth assays. Thus, allneurotrophins support the survival of embryonic neural crest-derivedsensory neurons. Survival of embryonic sympathetic neurons is onlysupported by NGF, while survival of placode-derived sensory neurons issupported by NT-3 and BDNF (Grotz et al., 1992). Survival of sensoryneurons of the dorsal root ganglion is supported by both NGF and BDNF.NT-3 elicits neurite outgrowth of sensory neurons from dorsal rootganglion, sympathetic chain ganglia, and nodose ganglion, as well assupports survival of nodose ganglia neurons and dorsal root ganglionneurons. Thus, neuronal survival assays or neurite outgrowth assays canbe run to determine the pantropicity of the pantropic neurotrophins.

[0132] Thus, neurotrophin specificity is determined by the neurotrophinreceptor binding, and the neuronal survival assays and/or neuriteoutgrowth assays. Thus, a pantropic neurotrophin with NGF specificitymeans a neurotrophin which exhibits at least the bindingcharacteristics, neuronal survival assay specificity, or the neuriteoutgrowth assay specificity of NGF. Similarly, a pantropic neurotrophinwith BDNF, NT-3 or NT-4/5 specificity exhibits at least the bindingcharacteristics, neuron survival assay specificity, or neurite outgrowthassay specificity of BDNF, NT-3 or NT-4/5, respectively.

[0133] In an additional embodiment, pantropic neurotrophins are made byconstructing covalent heterodimers. Normally, neurotrophins arehomodimers, comprising two identical monomers which are non-covalentlyassociated. In this embodiment, as outlined below, pantropicity isconferred by each monomer containing domains which confer differentneurotrophic specificity. Alternatively, pantropicity may be created bycovalently attaching two different neurotrophins with differentspecificities to create a covalent heterodimer. Thus, for example, a NGFmonomer may be covalently attached to a NT-3 monomer, resulting in apantropic neurotrophin with both NGF and NT-3 specificity. Similarly,covalent heterodimers may be made with any combination of NGF, NT-3,NT-4/5, BDNF or CNTF to create pantropic neurotrophins with at least twospecificities. In addition, this procedure may be done with monomerswhich are themselves pantropic, resulting in covalent dimers of anycombination of pantropic and single specificity monomers. Thus, apantropic covalent dimer may be a homodimer of two pantropic monomers.However, to be included within the definition of the present invention,the pantropic covalent dimer must have at least two, and preferablythree, neurotrophin specificities.

[0134] The covalent attachment is preferably done as a direct fusion ofthe nucleic acid, such that when the protein is expressed, theC-terminus of the first monomer is attached directly to the N-terminusof the second monomer, creating a single nucleic acid encoding thedimer. In alternative embodiments, a linker may be used, such as shortrepeats of glycine, or glycine and serine; for example, a linker such asgly-gly or gly-gly-ser-gly-gly may be used. This is done usingtechniques well known in the art. Other techniques for the covalentattachment of proteins are well known in the art.

[0135] Pantropic neurotrophins accomplish pantropic binding, or, asdiscussed above, pantropic neuronal survival, by containing domainswhich confer neurotrophin receptor specificity or binding. A domain maybe defined in one of two ways. In the first embodiment, a domain is aportion of the neurotrophin which confers some neurotrophic specificity.In this embodiment, a single monomer of the pantropic neurotrophincontains one or several domains which confer different specificities.The domains can range in size from a single amino acid to about 10-15amino acids. The domain may be comprised of a combination of amino acidsfrom a different neurotrophin than the host neurotrophin, i.e. a domainfrom one neurotrophin may be substituted into a second neurotrophin,conferring pantropicity to the second neurotrophin. Alternatively, thedomain may result from amino acid substitutions which are not based onhomology to existing neurotrophins, as outlined below. In the preferredembodiment, the domain comprises a continuous sequence of amino acids;that is, a single stretch of amino acids is replaced. In otherembodiments, the domain may be comprised of discontinuous amino acids;for example, several regions within the neurotrophin may conferspecificity, and thus replacements at several positions within theneurotrophin are necessary for pantropicity.

[0136] In some embodiments, there is more than one domain within aneurotrophin which can confer neurotrophic specificity, which willdepend on the particular neurotrophin. BDNF, for example, has a numberof domains which appear to confer BDNF specificity. The presentinvention shows that a single amino acid change in NT-3, from asparticacid at position 15 to an alanine, confers BDNF specificity to NT-3.This domain can also be imported into the NGF and NT-4/5 sequences atthe positions that correspond to position 15 in NT-3; i.e. position 16in NGF or position 18 in NT-4/5. It should be understood that thecorresponding amino acids are determined by an examination of thealignment of the sequences as depicted in U.S. Pat. No. 5,364,769. Inaddition to this domain, there are other domains within BDNF whichconfer BDNF specificity. For example, the substitution of the BDNFsequence-ffom positions 78 to 88 (QCRTTQSYVR), or from positions 93-99(SKKRIG) may confer BDNF specificity (55).

[0137] Similarly, NT-3 has a number of domains which may confer NT-3specificity when substituted into a different neurotrophin. A number ofresidues of NT-3 have been shown to be important in NT-3 trkC receptorbinding as well as bioactivity assays. Specifically, mutations atpositions R103, D105, K80, Q83, E54, R56, T22, Y51, V97, Y11, E7, R8,E10 and R68 all contribute to NT-3 specificity, since mutations at thesepositions in NT-3 cause decreases in NT-3 activity. Of these, K80, Q83,T22, and V97 are within variable regions, and the rest are found withinconstant regions. In addition, residues in the vicinity of the residuesmay also give NT-3 specificity. In some embodiments, changes in theconstant regions may also give NT-3 specificity. Alternatively,mutations at positions R31 and E92 caused increases in NT-3 binding;specifically, R31A and E92A NT-3 showed increased trkC binding. Thesemutations can be directly imported into neurotrophins besides NT-3,using the procedures described below. The amino acids at any of thesepositions may be changed, as outlined below.

[0138] NGF has a number of domains which may confer NGF specificity whensubstituted into a different neurotrophin. The N-terminal amino acids ofNGF confer NGF specificity when substituted for the N-terminal residuesof NT-3. Specifically, the 7 N-terminal amino acids (SSSHPIF) of NGF maybe substituted for the 6 N-terminal amino acids of NT-3 (YAEHKS),resulting in a pantropic NT-3 with NGF specificity. The exact number ofNGF N-terminal residues is not crucial; as shown in the Examples, andparticularly in Example 3, the histidine at amino acid position 4appears to be quite important for NGF specificity; thus from about 4 toabout 10 N-terminal residues may be exchanged although in someembodiments, a single amino acid change will be sufficient. Similarly, anumber of other residues of NGF have been shown to be important in NGFtrkA receptor binding as well as bioactivity assays. For example, thereare a number of residues which, when mutated, lose NGF activity. Thisshows the importance of the residue for NGF specificity. These residuesinclude, but are not limited to, H4, P5, V18, V20, G23, D30, Y52, R59,R69, H75, Y79, T81, and R103. Of these, D30, R59, Y79, and T81 are in“variable regions”, i.e. regions which vary between the differentneurotrophins, with the remainder in constant regions. In someembodiments, the variable region residues are more likely to cause NGFspecificity, since constant region residues may be important for generalstructure and characteristics, and may not confer specificity. However,as shown above for the D15A mutation, mutations in the constant regionscan confer specificity as well.

[0139] Furthermore, there are a number of amino acid substitutions inNGF which increase NGF binding and/or bioactivity. Accordingly, thesesubstitutions may be imported into other neurotrophin backbones toconfer NGF specificity. These residues include, but are not limited to,E11, F12, D24, E41, N46, S47, K57, D72, N77, H84, D105, and K115.

[0140] Once identified, the residues important in neurotrophinspecificity can be replaced by any of the other amino acid residuesusing techniques described in the examples and well-known techniques forsite-directed mutagenesis. Generally, the amino acids to be substitutedare chosen on the basis of characteristics understood by those skilledin the art. For example, when small alterations in the characteristicsare desired, substitutions are generally made as discussed above.

[0141] In the context of a covalent heterodimer, a domain may also referto the entire neurotrophin monomer. Thus, a pantropic covalentheterodimer can be comprised of a domain which confers NT-3 specificity,i.e. the NT-3 monomer, covalently attached to a domain that confers BDNFspecificity, i.e. the BDNF monomer. Similarly, an NT-3 monomer may bepaired with an NGF monomer, or an NGF monomer may be paired with a BDNFmonomer. In addition, covalent heterodimers may be made with NT-4/5 andCNTF monomers as well. In these embodiments, the domain is large, andgenerally comprises most or all of the wild-type neurotrophin amino acidsequence.

[0142] In one embodiment, the agonsit is a pantropic or chimeric NT-3.In this context, a pantropic NT-3 is a pantropic neurotrophin which hasan amino acid sequence homologous to the amino acid sequence of NT-3,with domains which confer other neurotrophin specificities. In thepreferred embodiment, the domains are substituted for NT-3 residues;that is, some number of amino acids are deleted from the NT-3 sequence,and an identical or similar number of amino acids are substituted,conferring an additional specificity. For example, the MNTS-1 (multipleneurotrophic specificities-1) pantropic NT-3 comprises the first 7 aminoacids of NGF replacing the 6 N-terminal residues of NT-3, plus the D15Asubstitution. The MNTS-1 pantropic NT-3 has NT-3, NGF, and BDNFspecificities, and also binds to the p75 receptor. Other pantropic NT-3sare made using minimal changes within the N-terminus. For example, sinceH4 and P5 are conserved among NGFs, and 2 hydrophobic residues inpositions 6 and 7 are conserved, the following variants are made: 1)YASHPIF-hNT-3; 2) YAHPIF-hNT-3; 3) YASHPIS-hNT-3; 4) YAEHPIF-hNT-3; 5)YAQHPIF-hNT-3. When the D1SA substitution is added, the resultingneurotrophins exhibit NGF, NT-3 and BDNF specificity. Alternatively,replacing the variable region 2 or 3 or 4, or combinations, of NT-3 withthe corresponding region from NGF gives a pantropic neurotrophin withboth NT-3 and NGF specificity. A pantropic NGF can be made with with aD16A substitution, which confers BDNF specificity, plus substitutions inthe pre-variable region 1 (V18E+V20L+G23T) and in variable region 4(Y79Q+T81K+H84Q+F86Y+K88R). Alternatively, the substitutions in thepre-variable region 1 can be made with only single amino acidsubstitutions in variable region 4; for example, V18E+V20L+G23T and oneof Y79Q, T81K, H84Q, F86Y, or K88R may be made.

[0143] In a preferred embodiment, the agonist is a chimeric or pantropicNT-4/5, preferably made with a trkC binding region. NGF specificity maybe conferred on NT-4/5 by replacing the N-terminal 9 amino acids ofNT-4/5 with the N-terminal 7 amino acids of NGF.

[0144] In one embodiment, binding to the p75 receptor by the pantropicneurotrophin has been substantially diminished or eliminated. Forexample, there are a variety of amino acid residues which contribute top75 binding, in which mutations result in diminished p75 binding. InNT-3, mutations at positions R68, Y11, K73, R114, K115, Y51, K73, R31and H33 and in NGF, mutations at positions F12, 131, K32, K34, K50, Y52,R69, K74, K88, L112, S113, R114, and K115 all result in diminished p75binding. Since F12, 131, K50, Y52, R69, and K74 are all within constantregions of the neurotrophins; these changes are expected to alter p75binding in the other neurotrophins as well. The other residues may bealtered as well.

[0145] In addition to the amino acid changes outlined above, thoseskilled in the art understand that some variability of the amino acidsequence is tolerated without altering the specificity andcharacteristics of the neurotrophin. Thus, pantropic neurotrophins canhave amino acid substitutions, insertions or deletions compared to thewild-type sequences which do not affect pantropicity but are merelyvariations of the sequence. In some embodiments, these mutations will befound within the same positions identified as important to specificity;i.e. in some cases, neutral mutations may be made without changingneurotrophin specificity.

[0146] The pantropic neurotrophins of the present invention can be madein a variety of ways, using recombinant technology as discussed above.In a preferred embodiment, the pantropic neurotrophins of the inventionare expressed in mammalian cells. Mammalian expression systems are alsoknown in the art. In one embodiment, pantropic neurotrophins areproduced in yeast cells. Yeast expression systems are well known in theart, and include expression vectors for Saccharornyces cerevisiae,Candida albicans and C. maltosa, Hansenula polymorpha, Kluyverornycesfragilis and K. lactis, Pichia guillerimondii and P. pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica. The methods ofintroducing exogenous nucleic acid into yeast hosts, as well as otherhosts, is well known in the art, and will vary with the host cell used.In a preferred embodiment, pantropic neurotrophins are expressed inbacterial systems. Expression vectors for bacteria are well known in theart, and include vectors for Bacillus subtilis, E. coli, Streptococcuscremoris, and Streptococcus lividans, among others. The bacterialexpression vectors are transformed into bacterial host cells usingtechniques well known in the art, such as calcium chloride treatment,electroporation, and others. In one embodiment, pantropic neurotrophinsare produced in insect cells. Expression vectors for the transformationof insect cells, and in particular, baculovirus-based expressionvectors, are well known in the art. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form; for example the “MaxBac” kit from Invitrogen in San Diego.Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for Aedes aegypti, Autographacalifornica, Bombyx mori, Drosophila melangaster, Spodoptera frugiperda,and Trichoplusia ni.

[0147] Once expressed, chimeric or pantropic neurotrophins are used asneurotrophic factors. These chimeric or pantropic neurotrophins may beutilized in various compositions, assays, and therapeutic applicationsof the invention For use in the assays of the invention the agonist canbe labeled. By “labeled” herein is meant an agonist that has at leastone element, isotope or chemical compound attached to enable thedetection of the neurotrophin bound to a neurotrophin receptor. Ingeneral, labels fall into three classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) immune labels, which may beantibodies or antigens; and c) colored or fluorescent dyes. The labelsmay be incorporated into the neurotrophin at any position. Oncelabelled, the neurotrophins are used to detect neurotrophin receptors,either in vitro or in vivo. For example, the presence of neurotrophinreceptors can be an indication of the presence of certain cell types,useful establishing and in scoring the assays. That is, a subpopulationof certain cell types may be shown by the binding of the labeledneurotrophin to the cells via the receptors.

[0148] Additionally, the neurotrophins are useful as standards in assaysof the invention. For example, the activity of a variant neurotrophin inany particular assay may be determined using known neurotrophinstandards, and then the variant neurotrophin may be used in thediagnosis and quantification of neurotrophins and other agonists.

[0149] As will be understood by those skilled in the art, the pantropicneurotrophins of the present invention can replace other neurotrophicfactors which are used as media components in the cultures as taughtherein and in teh methods of treatment taught herein. The amount of thepantropic neurotrophins to be added can be easily determined usingstandard assays.

[0150] Purification of Agonists

[0151] Techniques used for separating the agonist from impurities dependon which particular agonist is being employed. These procedures mayinclude, for example, one or more steps selected from immunoaffinitychromatography, ion-exchange column fractionation (e.g., on DEAE ormatrices containing carboxymethyl or sulfopropyl groups), chromatographyon Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentillectin-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, ButylToyopearl, Phenyl Toyopearl, or protein A Sepharose, SDS-PAGEchromatography, silica chromatography, chromatofocusing, reverse phaseHPLC (e.g., silica gel with appended aliphatic groups), gel filtrationusing, e.g., Sephadex molecular sieve or size-exclusion chromatography,chromatography on columns that selectively bind the trkB or trkCagonist, such as trkB or trkC receptors or antibody-affinity, andethanol or ammonium sulfate precipitation. A protease inhibitor may beincluded in any of the foregoing steps to inhibit proteolysis. Examplesof suitable protease inhibitors include phenylmethylsulfonyl fluoride(PMSF), leupeptin, pepstatin, aprotinin,4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride-bestatin,chymostatin, and benzamidine.

[0152] Therapeutic Compositions and Administration of Agonists Agoniststo trkB or trkC alone, in combination with each other, or optionally incombination with ototoxic pharmaceuticals, are believed to find use asdrugs for in vivo treatment of mammals, ex viuo treatments involvingtransplant or assays involving organs such as during perfusion, and invitro assays and screening methods. For example, the trkB or trkCagonist alone or in combination with each other will be useful intreating balance impairments in cases where pharmaceutical drugs arelimited in their dosage or display side-effect of a oto-neurologicalbalance impairment.

[0153] In the preferred embodiment, the neurotrophin(s) is administeredto a patient to treat neural-related (associated with neurondegeneration, damage or loss) imbalance impairment, prophylactically ortherapeutically. Preferably hair cell loss or damage is not present ornot at a significant level that would hinder balance recovery. Specificexamples include, but are not limited to neuropathies, and otherconditions characterized by necrosis, damage, or loss of neuronsaffecting balance, whether caused by trauma, injury, aging, noise,environmental toxins, or ototoxic pharmaceutical drugs. For example,neuropathies associated with certain conditions such as diabetes, AIDS,or chemotherapy may be treated using the compositions and methods of thepresent invention.

[0154] Therapeutic formulations of agonist(s) (and optionally ototoxicpharmaceutical drug) for treating balance impairments are prepared forstorage by mixing the agonist(s) or drug having the desired degree ofpurity with optional physiologically acceptable carriers, excipients, orstabilizers (Remington's Pharmaceutical Sciences, 16th edition, Oslo,A., Ed., [1980]), in the form of lyophilized cake or aqueous solutions.Acceptable carriers, excipients, or stabilizers are non-toxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins-chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween, Pluronics, or polyethylene glycol (PEG).

[0155] The agonist(s) are also suitably linked to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. Nos.4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0156] The agonist(s) to be used for in viuo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution. The agonist(s) ordinarily will be stored in lyophilizedform or in solution. Preferably, it is free or substantially free (atleast 80%, preferably at least 90%, more preferably at least 95%, andeven more preferably at least 99% pure) of contaminating polypeptidesfrom the purification source.

[0157] Therapeutic agonist compositions generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0158] The agonist(s) is administered in an acute or chronic fashion, asmay be required, for prophylactic and therapeutic applications, by anumber of routes including: injection or infusion by intravenous,intraperitoneal, intracerebral, intramuscular, intradermally,intraocular, intraarterial, subcutaneously, or intralesional routes,topical administration, orally if an orally active small molecule isemployed, using sustained-release systems as noted below, or by anindwelling catheter using a continuous administration means such as apump, by patch, or implant systems, e.g., intracerebral implantation ofa sustained-release vehicle. Agonist(s) is administered continuously byinfusion or by periodic bolus injection if the clearance rate issufficiently slow, or by administration into the blood stream, lymph,CNS or spinal fluid. A preferred administration mode is directly to theaffected portion of the ear or vestibule, topically, and, preferably tothe affected neurons, so as to direct the molecule to the source andminimize side effects of the agonists.

[0159] Neurotrophin, preferably NT-4/5, can be injected throughchronically implanted cannulas or chronically infused with the help ofosmotic minipumps. Subcutaneous pumps are available that deliverproteins through a small tubing to the appropriate area. Highlysophisticated pumps can be refilled through the skin and their deliveryrate can be set without surgical intervention. Examples of suitableadministration protocols and delivery systems involving a subcutaneouspump device or continuous infusion through a totally implanted drugdelivery system are those used for the administration of dopamine,dopamine agonists, and cholinergic agonists to Alzheimer patients andanimal models for Parkinson's disease described by Harbaugh, J. NeuralTransm. Suppl., 24: 271-277 (1987) and DeYebenes et al., Mov. Disord.,2: 143-158 (1987), the disclosures of which are incorporated herein byreference. It is envisioned that it may be possible to introduce cellsactively producing agonist into areas in need of increasedconcentrations of agonist.

[0160] Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) asdescribed by Langer et al., J. Biomed. Mater. Res., 15: 167-277 [1981]and Langer, Chem. Tech., 12: 98-105 [1982] or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 [1983]), non-degradable ethylene-vinyl acetate (Langer etal., supra), degradable lactic acid-glycolic acid copolymers such as theLupron Depot™ (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid (EP 133,988). The agonist(s) also may be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-[methylmethacylate] microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Osol, A., Ed.,(1980).

[0161] While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release proteins for shorter time periods. Whenencapsulated proteins remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for proteinstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

[0162] Sustained-release agonist(s) compositions also includeliposomally entrapped agonist(s). Liposomes containing agonist(s) areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Pat.Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomesare of the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. % cholesterol, the selectedproportion being adjusted for the optimal agonist therapy.

[0163] A specific example of a suitable sustained-release formulation isin EP 647,449.

[0164] An effective amount of agonist(s) to be employed therapeuticallywill depend, for example, upon the therapeutic objectives, the route ofadministration, the species of the patient, and the condition of thepatient. Accordingly, it will be necessary for the therapist to titerthe dosage and modify the route of administration as required to obtainthe optimal therapeutic effect. As is known in the art, adjustments forage as well as the body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art. A typical daily dosage of TrkB or trkC agonistused alone might range from about 1 μg/kg to up to 100 mg/kg of patientbody weight or more per day, depending on the factors mentioned above,preferably about 10 μg/kg/day to 10 mg/kg/day. Typically, the clinicianwill administer agonist until a dosage is reached that repairs,maintains, and, optimally, reestablishes neuron function to relieve theimbalance impairment.

[0165] Generally, the agonist is formulated and delivered to the targetsite at a dosage capable of establishing at the site an agonist levelgreater than about 0.1 ng/ml, more typically from about 0.1 ng/ml to 5mg/ml, preferably from about 1 to 2000 ng/ml. In a specific embodimentof the invention, a pharmaceutical composition effective in promotingthe survival of VGNs may provide a local neurotrophin proteinconcentration of between about 1 and 100 ng/ml, preferably 5 to 25ng/ml, and more preferably, between 10 and 20 ng/ml. The progress ofthis therapy is easily monitored by conventional assays and neurologicaldiagnostic methods.

[0166] If two agonists are administered together, they need not beadministered by the same route, nor in the same formulation. However,they can be combined into one formulation as desired. In a preferredembodiment NT-4/5 optionally is combined with or administered in concertwith or formed as a pantropic neurotrophin with a neurotrophic agonistto trkC. Both agonists can be administered to the patient, each ineffective amounts, or each in amounts that are sub-optimal but whencombined are effective. Preferably such amounts are about 10 μg/kg/dayto 10 mg/kg/day of each. In another preferred embodiment, theadministration of both agonists is by injection using, e.g., intravenousor subcutaneous means, depending on the type of agonist employed.Typically, the clinician will administer the agonist(s) until a dosageis reached that achieves the desired effect for treatment of the balanceimpairment. The progress of this therapy is easily monitored byconventional assays.

[0167] The two types of agonists, if used together, may be formulatedtogether in an appropriate carrier vehicle to form a pharmaceuticalcomposition that preferably does not contain cells. In one embodiment,the buffer used for formulation will depend on whether the compositionwill be employed immediately upon mixing or stored for later use, sincelong-term storage may bring into issue stability such as solubility andaggregation that can be addressed by altering the pH. The finalpreparation may be a stable liquid or lyophilized solid.

[0168] The agonist(s) optionally is combined with or administered inconcert with ototoxic pharmaceutical drugs. Initially the drugs areadministered in conventional therapies known for the ototoxicpharmaceutical. Adjustments to the therapies are at the discretion ofthe skilled therapist to titrate dosages and conditions that decreaseototoxicity-related imbalance while maintaining, and preferablyimproving, treatment outcomes with the ototoxic pharmaceutical drug.

[0169] Accordingly, methods for preventing or reducing ototoxicity of anaminoglycoside antibiotic or other ototoxic pharmaceutical are disclosedherein, which comprise the administration of an effective dose of a trkBor trkC agonist. In addition, provided herein are compositions havingreduced ototoxicity as a result of incorporation of theototoxicity-inhibiting trkB or trkC agonists of the present invention.These pharmaceutical compositions comprise an effectiveototoxicity-inhibiting amounts of agonists as described herein,therapeutically effective amounts of the ototoxic pharmaceutical drug,e.g. aminoglycosides antibiotic, anti-neoplastic agent such ascisplatin, and optionally a pharmaceutically acceptable carrier and/orvehicle which would be familiar to one skilled in the pharmaceuticalarts. The actual amounts of ototoxic pharmaceutical drug employed willrange from those given in standard references for prescription drugs,e.g. “Physicians Desk Reference” (1995), “Drug Evaluations” AMA, 6thEdition (1986); to amounts somewhat larger since the ototoxicitypotential is reduced in these compositions.

[0170] The effective amounts of such agents, if employed, will be at thephysician's or veterinarian's discretion. Dosage administration andadjustment is done to achieve the best management of imbalance (and whenused in conjunction with an ototoxic pharmaceutical drug, the indicationfor the ototoxic drug). The dose will additionally depend on suchfactors as the type of drug used and the specific patient being treated.Typically the amount employed will be the same dose as that used if thedrug were to be administered without agonist; however, lower doses maybe employed depending on such factors as the presence of side-effects,the condition being treated, the type of patient, and the type ofagonist and drug, provided the total amount of agents provides aneffective dose for the condition being treated. For example, a test dosemay be 5 mg, which is then ramped up to 10-20 mg per day, once a day, to25 mg twice per day (BID) or three times per day (TID), and may betitrated to 50 mg BID or TID as the patient tolerates it. Tolerancelevel is estimated by determining whether decrease in imbalanceimpairment is accompanied by signs of observed side-effects. Adiscussion of the dosage, administration, indications andcontraindications associated with ototoxic pharmaceuticals optionallyused with the neurotrophins in the methods of the invention can be foundin the Physicians Desk Reference, Medical Economics Data Production Co.,Montvale, N.J. (1995).

[0171] In preferred embodiments therapeutic formulations contain NT-4/5,a fragment, variant, or pantropic, and can be prepared for storage bymixing NT-4/5 having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences, supra,) in the form of lyophilizedcake or aqueous solutions.

[0172] The compositions herein also may suitably contain other peptidegrowth factors, most preferably hair cell growth factors, perhapsretinoic acid. Such growth factors are suitably present in an amountthat is effective for the purpose intended, i.e., to promote restorationor recovery of hair cells when desired, or to enhance growth or recoveryof neurons. Although the present results indicate that particularneurotrophins have strong protective effects on VGNs, they did notprotect hair cells from the ototoxic drugs. If hair cell loss due toototoxicity is significant, balance impairment recovery could beimproved by new hair cell growth or regeneration. Recent studies havesuggested possible candidates (Forge et al., 1993; Cotanche and Lee,1994; Tsue et al., 1994a; Cotanche and Lee, 1994; Kelley et al., 1995).For example, diffusible factors such as TGF-α and EGF (Lambert, 1994;Yamashita and Oesterle, 1995) or components derived from antibiotictreated inner ear tissue (Tsue et al., 1994b) stimulate proliferation ofsupporting cells. Retinoic acid alone or in combination with TGF-αfacilitates hair cell regeneration in vitro (Lefebvre et al., 1993,1995). As taught herein, neurotrophins will provide for prevention ofneuronal cell death after injury or insult by ototoxins.

[0173] The effectiveness of treating balance impairments with themethods of the invention can be evaluated by the following signs ofrecovery, including recovery of normal balance function, which can beassessed by known diagnostic techniques including those discussedherein, and normalization of nerve conduction velocity, which isassessed electrophysiologically.

[0174] In another embodiment, agonist compositions of the invention areused during clinical utricle implants to keep or improve viability ofvestibular ganglion neurons. Preferably a combination of a trkB and atrkC neurotrophin and a hair cell growth factor(s) will be used, eitheralone or in combination with a utricle implant.

[0175] Utricle Explants

[0176] In one embodiment of the invention is provided a method ofassaying for a trkB or trkC agonist that provides vestibular ganglionneuron protection or survival from an ototoxin. The assay steps includeculturing a utricle explant, administering a trkB or trkC agonist to theculture, administering an ototoxin to the culture, and determining theamount of protection or survival compared to a control culture to whichthe trkB or trkC agonist was not administered.

[0177] In a preferred embodiment of the invention is provided anorganotype utricle explant culture that utilizes a 3-D collagen matrixcultures and maintains its normal, in vivo architecture to provide avestibular assay system. The vestibular ganglion remain attached. Theexplant is cultured in three-dimensional (“3-D”) collagen gel in serumfree medium.

[0178] Embedding the utricle explants in the 3-D collagen was better formaintaining their normal architecture than floating the explants orplacing the explants on a monolayer substrate, since the explant tissuecould be kept unfolded and cell migration out of the tissue could belimited. By using neurofilament (N52) and phalloidin-FITC conjugatedouble labeling, the integrity of VGNs and the hair cells in the utriclewas demonstrated. Utricle explants prepared according to the inventionmaintained normal architecture in the 3-D collagen gel cultures asobserved by Nomarski micrographs of utricle tissue dissected from P3rats and grown for 2 days in vitro at low and high magnifications. TheVGNs and hair cells in the explants grew well and maintained theirnormal connectivity. The VGNs and hair cells remained in their normallocations. No gross cell death of VGNs and hair cells occurred underthis culture condition. Organotypic cultures of postnatal utricleexplants provided herein, in which the innervation of hair cells byvestibular neurons are intact, are useful to examine ototoxicity ofdifferent classes of ototoxins, including ototoxic pharmaceutical drugs,for example, salicylate, gentamicin, and cisplatin, and to search for ortest candidate agents that protect against the ototoxic effect. Todetermine if an ototoxin is able to induce degeneration of VGNs and/orhair cells in the utricle explant cultures, the ototoxin is added atdifferent concentrations to the culture after allowing the culture torecover from the in vitro explant. Cell count of remaining hair cellsand VGNs can be done to determine and quantify ototoxic effect. Sincethe density of the axons of VGNs is a reliable index of the number ofsurviving VGNs, in one embodiment the number of the VGN axons from agiven length (100 μm) in the middle of the utricle is counted.Phalloidin-labeled hair cells were also counted in the same way.

[0179] Organotypic culture of utricle explants offers advantages toexplore the mechanism of actions of ototoxins, to discover protectiveagents, and to search for hair cell growth factors, as it keeps theafferent neuronal innervation of hair cells intact and appears to followclosely the normal development pathway. According to the presentinvention, provided herein is a reliable, rapid, and facile method oftesting the effects of ototoxic agents and the drugs that prevent,reduce or treat these ototoxic effects. As exemplified herein anorganotypic culturing of postnatal cochlear explants in a 3-D collagenmatrix in well defined, serum-free medium provides these advantageswithout the need for a cumbersome Maximov slide assembly (Sobkowicz etal., 1975; Rastel et al., 1993) or undefined medium. Embedding theutricle explants in the 3-D collagen was better for maintaining thenormal architecture than floating the explants or placing the explantson a monolayer substrate, since the explant tissue could be keptunfolded and cell migration out of the tissue could be limited.

[0180] The following examples serve to more fully describe the manner ofusing the above-described invention. The following examples are offeredby way of illustration and not by way of limitation. The disclosures ofall citations in the specification are expressly incorporated herein byreference.

EXAMPLES Example I NT-4/5, BDNF and NT-3, but not NGF, Enhance VGNSurvival in Culture

[0181] The effects of trkB or trkC agonists to enhance neuronal survivalwas determined using VGNs in cell culture. VGN cell culture was preparedas follows. Vestibular ganglia were dissected from postnatal day 5 (P5)Wistar rats and were incubated in a mixture of 0.125% trypsin and 0.125%collagenase for 25 min at 37° C. The enzyme was inactivated with amixture of 0.005% soybean trypsin inhibitor (Sigma) and 0.005% DNase(Worthington) before trituration with 0.05% DNase in Eagle's BasalMedium (“BME”). After the undissociated tissues were separated byfiltering with a Nylon meshwork (33 μm in pore size), the dissociatedcells were preplated on a 35 mm untreated tissue culture dish for 25 minto enrich the neuronal population. Under these experimental procedures,about 3-5% of the cell population were vestibular ganglion neurons(VGNs) as determined by immunocytochemistry with a monoclonal antibody(N52; Boehringer) against neurofilament (200 kd). The cell suspensionwas finally plated on polylysine (500 μg/ml)Aaminin (20 μg/ml) coated16-well LabTek slides in 200 μl of serum-free medium (BME plusserum-free supplement (Sigma I-1884), 1% BSA, 2 mM glutamine, and 5mg/ml glucose) containing no antibiotics. Cells were plated at a densityof 100,000/well.

[0182] All human recombinant neurotrophins (obtained from Genentech,Inc.) were added to the cultures at the time of plating.

[0183] Cell counts and data analysis were performed as follows. After 2days in culture, viable VGNs were identified by labeling with theneurofilament monoclonal antibody N52 and counted using a grid ocularreticule covering an area of 1 mm² under a Zeiss Axiophot microscope.For each culture, about 10 randomly selected fields were counted. Inthese experiments, cell counts were performed in the same way forcontrol cultures. Data were collected from triplet cultures for each ofthe experimental groups. Data were then normalized as a percentage ofthe viable VGNs in the control cultures in each of the experiments.

[0184] Immunohistochemistry and immunocytochemistry were performed asfollows. For double antibody labeling, vestibular ganglia were dissectedfrom P5 rats and immersed immediately in 4% paraformaldehyde (in 0.1Mphosphate buffer, pH 7.4) for 1 hr. After the vestibular ganglia werecryoprotected with a 30% sucrose solution, cross sections were cut on acryostat. The sections were first blocked with a 10% normal goat serumin 1% triton-X 100 in phosphate buffered saline (PBS) for 20 min andthen incubated with a mixture of the monoclonal antibody N52 againstneurofilament 200 kD (5 μg/ml) and a rabbit antibody against theextracellular domain of trkB (anti-trkB₂₃₋₃₆, 2 μg/ml; Yan et al., 1994;Gao et al., 1995), a trkA antiserum (1:10,000, Clary et al., 1994), oran antiserum against p75 (1:10,000, Weskamp and Reichardt; 1991) in PBScontaining 3% normal goat serum and 1% Triton-X 100 overnight at 4° C.FITC-conjugated goat anti-mouse and Texas red-conjugated goatanti-rabbit secondary antibodies (1:70-100; Cappel) were then used toreveal the double labeling pattern on the sections of vestibularganglion. For horse radish peroxidase-mediated neurofilamentimmunostainings, VGN cultures were fixed in 4% paraformaldehyde (in 0.1Mphosphate buffer, pH 7.4) for 30 min, washed in PBS (pH 7.4), and theimmunostainings were performed using a biotinylated sheep anti-mousesecondary antibody and a streptavidin-horse radish peroxidase conjugate(1:200, Amersham Life Science), as described elsewhere.

[0185] The effect of neurotrophins on cell survival was determined onVGNs which were dissociated from postnatal day 5 (P5) rat vestibularganglia and plated in defined serum-free medium. Neurofilament proteinimmunocytochemistry using bright field microscopy was performed in theVGN cultures in serum-free medium in the absence or presence of 10 ng/mlof NT-4/5. In control cultures in serum-free medium withoutneurotrophins, about 35% of VGNs survived after 2 days. When NT-4/5 wasadded to the culture, an up to 2-fold increase was seen in the number ofneurons that survived, as identified by immunocytochemistry with amonoclonal antibody (N52) against neurofilament protein (200 kd), whichlabeled intensely both somata and processes of VGNs in the culture aswell as in the vestibular ganglion (see below, FIG. 3). Thesurvival-promoting effects of NT-4/5 on cultured VGNs weredose-dependent (FIG. 1). When different doses of NT-4/5 ranging from 0.1to 50 ng/ml were examined, a maximal effect was seen at a concentrationof 10 ng/ml.

[0186] Effects of other neurotrophins including NGF, BDNF, and NT-3,were also examined and compared to that of NT-4/5 on cultured VGNs at aconcentration of 10 ng/ml (FIG. 2). BDNF was equivalent to NT-4/5 inpromoting survival of VGNs (p<0.001 for both NT-4/5 and BDNF, ascompared to control). NT-3 displayed a significant effect (p<0.01) butwas less potent than NT-4/5 and BDNF (p<0.05). In contrast, NGF did notshow any detectable effect in VGN cultures. To determine whether therewere additive effects of the neurotrophins on VGN survival, NT-4/5 wascombined with BDNF or NT-3 at a dose of 10 ng/ml; however, nosynergistic effects were observed (FIG. 2).

[0187] When other growth factors including epidermal growth factor(EGF), basic fibroblast growth factor (βFGF), and insulin-like growthfactor-1 (IGF-1) were added to the cultures at the time of plating at aconcentration of 10 ng/ml, the number of surviving VGNs was not affected(FIG. 2), suggesting a selective response of VGNs to the threeneurotrophins, NT-4/5, BDNF and NT-3.

Example II TrkB-IgG and trkC-IgG Abolish Specifically theSurvival-Promoting Effects of NT-4/5, BDNF and NT-3

[0188] TrkB-IgG and trkC-IgG have been previously reported to bespecific antagonists for NT-4/5 (or BDNF) and NT-3, respectively(Shelton et al., 1995). To confirm that the survival-promoting effectsof NT-4/5, BDNF and NT-3 were specific, trkB-IgG or trkC-IgG fusionprotein was added to the culture along with NT-4/5, BDNF or NT-3. Allhuman recombinant neurotrophins (Genentech, Inc.) were added to thecultures at the time of plating. To block the effects of theneurotrophins, 1 μg/ml of trkB-IgG or trkC-IgG (kindly provided by Dr.Dave Shelton), specific antagonists for NT-4/5 (and BDNF) or NT-3,respectively (Shelton et al., 1995), was added to some of theexperimental cultures at the same time when neurotrophins were added.

[0189] As shown in FIG. 3, the presence of the trkB-IgG fusion proteincompletely abolished the survival-promoting effects of NT-4/5 or BDNF,but not that of NT-3. Similarly, addition of trkC-IgG inhibited theactivity of NT-3 but not that of NT-4/5 or BDNF (FIG. 3). While trkB-IgGand trkC-IgG together blocked the effects of NNT-4/55 and NT-3, bythemselves they did not show detectable effects in the VGN cultures.These results confirm the specificity of trkB-IgG or trkC-IgG andsupport the idea that the survival-promoting effects observed with trkBor trkC agonists NT-4/5, BDNF or NT-3 are specific.

Example III VGNs make trkB and p75 Proteins, but not trkA Protein

[0190] To determine which neurotrophin receptors are expressed by VGNs,immunohistochemistry with antisera against trkB (Yan et al., 1994; Gaoet al., 1995), trkA (Clary et al., 1994) and p75 (Westkamp andReichardt, 1991) was performed. Dual immunohistochemistry on crosssections of the vestibular ganglion with trkB, trkA, or p75 andneurofilament protein antibodies was performed. Texas red microscopy wasused to show the staining pattern of antibodies against trkB, trkA andp75, respectively, while fluorescent microscopy was used to show theimmunostainings of neurofilament antibody (N52) in the same sections.N52 clearly stained somata and processes of VGNs. Note that while VGNsomata and processes were heavily labeled by trkB and p75 antibodies,the staining by trkA antiserum was absent. While trkB antiserum labeledboth the somata and processes of VGNs in the vestibular ganglion, trkaantiserum failed to detect the presence of trkA protein in theseneurons. When the cross sections of vestibular ganglion weredouble-labeled with a monoclonal antibody (N52) against neurofilamentprotein (200 Kd) which stains all processes and cell bodies of the VGNs,all neurofilament-positive VGNs were also immunoreactive to antiserumagainst trkB, but not to antiserum against trkA. In addition, a majorityof VGNs were double labeled by an antiserum against p75, thelow-affinity receptor for all neurotrophins. These results indicate thatVGNs produce trkB and p75 proteins, but not trkA protein.

Example IV NT-4/5, BDNF and NT-3 Protect VGNs against Neurotoxicity ofOtotoxic Drugs

[0191] The ability of trkB or trkC agonists to protect neurons fromototoxicity was determined using VGNs in cell culture. When cisplatin orgentamicin was added to the culture, a dose-dependent inhibition of VGNsurvival was observed (FIGS. 4 and 5). At a dose of 4 μg/ml of cisplatinor 3 mgiml of gentamicin, a majority of VGNs died in the culture. Allhuman recombinant neurotrophins (Genentech, Inc.) were added to thecultures at the time of plating. To block the effects of theneurotrophins, 1 μg/ml of trkB-IgG or trkC-IgG (kindly provided by Dr.Dave Shelton), specific antagonists for NT-4/5 (and BDNF) or NT-3,respectively (Shelton et al., 1995), was added to some of theexperimental cultures at the same time when neurotrophins were added. Insome experiments, cisplatin (Bristol-Myers Squibb) or gentamicin sulfate(Sigma) was added at various concentrations at the time of plating,either alone or in combination with 10 ng/ml of different neurotrophins.In other experiments, epidermal growth factor (EGF), basic fibroblastgrowth factor (βFGF) and insulin-like growth factor-1 (IGF-1) were addedto the culture at the time of plating at a concentration of 10 ng/ml.Cell counts and data analysis were performed as described in the aboveExamples except that in some cultures in which high concentrations ofcisplatin or gentamicin were added, cell counts were made from theentire area of the culture wells because the overall number of viableVGNs was small. In these experiments, cell counts were performed in thesame way for control cultures.

[0192] To examine whether neurotrophins could protect the VGNs fromcisplatin neurotoxicity, neurotrophins were added together with 3different doses of cisplatin. At 1 μg/ml or 2μg/ml of cisplatin, thenumber of surviving VGNs in the culture containing NT-4/5 was not onlyhigher than that in the culture with cisplatin alone, but also higherthan the control culture without cisplatin, indicating that NT-4/5prevents the VGN from cisplatin neurotoxicity and promotes VGN survival.At 4 μg/ml of cisplatin, NT-4/5 still significantly ameliorated thetoxic effects of cisplatin (p<0.001) and protected VGNs from cell death,although it was about 60% of the survival levels of control cultures inthe absence of cisplatin (FIG. 4). An equivalent effectiveness of BDNFwas observed against cisplatin neurotoxicity. Significant protective,but less potent effects were seen for NT-3. In contrast, NGF exhibitedno protective effects (FIG. 4). Similar protective effects of NT-4/5,BDNF and NT-3 on VGNs were observed against neurotoxicity of gentamicin.As seen in the experiments with cisplatin, while NGF showed no effect,BDNF displayed an effect equivalent to NT-4/5, and NT-3 also exhibitedsignificantly protective effect (FIG. 5). Finally, no additive effectswere observed when NNT-4/55 was combined with BDNF or NT-3 in theculture containing cisplatin or gentamicin (FIG. 4 and FIG. 5).

Example V Utricle Explant Cultures

[0193] An organotype utricle explant culture that utilizes a 3-Dcollagen matrix cultures and maintains its normal, in vivo architecturewas prepared to provide a vestibular system. The utricle was dissected,with the vestibular ganglion attached, from P3 Wistar rats and culturedin three-dimensional (“3-D”) collagen gel in serum free medium. In oneembodiment a droplet (20 pi) of freshly made collagen gel which wasplaced on the bottom of a 35 mm Nunc tissue culture dish, modified fromwhat described previously (Gao et al., 1991) as follows. Rat tailcollagen (type I, Collaborative Research) was mixed with 10× BME mediumand 2% sodium carbonate in a ratio of 10:1:1 and placed on ice justbefore use. The collagen matrix containing the utricle explant wasincubated at 37° C. for 5-10 min until gellation. The matrix was thencultured in defined serum-free medium using sufficient medium to coverthe explant (2 ml of serum-free medium (BME plus serum-free supplement(Sigma I-1884), 1% BSA, 2 mM glutamine, and 5 mg/ml glucose; containingno antibiotics) The culture medium was changed every other daythereafter.

[0194] Embedding the utricle explants in the 3-D collagen was better formaintaining their normal architecture than floating the explants orplacing the explants on a monolayer substrate, since the explant tissuecould be kept unfolded and cell migration out of the tissue could belimited. By using neurofilament (N52) and phalloidin-FITC conjugatedouble labeling, the integrity of VGNs and the hair cells in the utriclewas demonstrated. Utricle explants prepared according to the inventionmaintained normal architecture in the 3-D collagen gel cultures asobserved by Nomarski micrographs of utricle tissue dissected from P3rats and grown for 2 days in vitro at low and high magnifications. TheVGNs and hair cells in the explants grew well and maintained theirnormal connectivity. The VGNs and hair cells remained in their normallocations. No gross cell death of VGNs and hair cells occurred underthis culture condition.

Example VI Ototoxicity in Utricle Explant Cultures and ProtectiveEffects Of Neurotrophins In Utricle Explant Cultures

[0195] Organotypic cultures of postnatal utricle explants providedherein, in which the innervation of hair cells by vestibular neurons areintact, are useful to examine ototoxicity of different classes ofototoxins, including ototoxic pharmaceutical drugs, for example,salicylate, gentamicin, and cisplatin, and to search for or testcandidate agents that protect against the ototoxic effect. To determineif an ototoxin is able to induce degeneration of VGNs and/or hair cellsin the utricle explant cultures, the ototoxin was added at differentconcentrations to the culture after allowing the culture to recover fromthe in vitro explant. Recovery typically occurred after two days.Histochemical double-labeling of the utricle explant cultures with aneurofilament antibody (e.g., Texas red-mediated; N52) and phalloidin(e.g., FITC-conjugated) were used to compare control cultures(untreated) with cultures treated with the ototoxin. While theneurofilament antibody (Texas red-mediated) labeled the VGNs, thephalloidin-FITC conjugate stained the hair cells. Typically threecultures per experimental paradigm were studied in each individualexperiment. Three or more separate repetitions of the experiment wereconducted to validate the ototoxic effect. Cell count of remaining haircells and VGNs were performed. Since the density of the axons of VGNsappeared to be a reliable index of the number of surviving VGNs, thenumber of the VGN axons from a given length (100 μm) in the middle ofthe utricle was counted for different experimental groups and plotted.Phalloidin-labeled hair cells were also counted in the same way. In someof the experiments, improved accuracy was obtained by using serialcryostat sections (10 micrometer in thickness) for VGNs. Every fifthsection was collected on one slide and stained with cresyl violet. Totalnumber of remaining VGNs was determined as five-times the number of VGNscounted on the slide.

[0196] Neurotrophins are members of the NGF family of proteins. Theyhave been widely shown to regulate the differentiation and survival ofdeveloping neurons (Korsching, 1993, Gao et al., 1995a) as well as toaid in the repairing or recovery of adult CNS neurons from injury andtoxins (Hefti, 1986; Knusel et al., 1992; Yan et al, 1992; Gao et al.,1995b). They exert their biological functions through activation ofhigh-affinity binding receptors, the trks with high characteristicspecificity (Barbacid, 1993; Snider, 1994). As reported herein, VGNsexpress specific trk proteins. Hair cells express certain neurotrophingenes (Pirvola et al., 1992; Schecterson and Bothwell, 1994; Wheeler etal., 1994). In dissociated cell culture systems, as shown herein,specific neurotrophins promote survival of VGNs. As demonstrated hereinneurotrophins protect VGNs from cisplatin ototoxicity. Similarly, asdemonstrated for the first time herein, these neurotrophins also protectvestibular ganglion neurons from gentamicin in vitro. Of the fourneurotrophins tested, NGF is ineffective. NT-4/5 and BDNF areequivalently and potently effective, and NT-3 displays a less potent butsignificant survival-promoting effect. The TrkB and TrkAimmunohistochemistry on the sections of vestibular ganglia correlatesstrongly with the differential survival-promoting effects of NT-4/5,BDNF and NGF in VGN cultures. While the absence of immunostaining ofTrkA antiserum is in agreement with the negative results of NGF on theseneurons, the presence of TrkB protein in the VGNs in the presentexperiment suggests a direct action of NT-4/5 and BDNF on the VGNs.Consistent with these results, other growth factors including EGF, bFGFand IGF-1 show no detectable effects. The fact that addition of TrkB-IgGand TrkC-IgG to the culture specifically blocks the effects of NT-4/5,BDNF and NT-3 in the present study provides additional support for thenotion of direct action of the three neurotrophins on VGNs. Given thatthe survival potency of NT-3 is lower than that of NT-4/5 and BDNF, itis quite possible that while most VGNs express TrkB gene, only a subsetof these neurons co-express TrkC gene. The substantial loss of VGNs thathas been reported for the TrkB knockout mice but not for the micelacking the TrkC gene (Fritzsch et al., 1995) supports this notion.

[0197] Gentamicin, an aminoglycoside commonly used for treating diseasescaused by Gram-negative bacteria, has unfortunate side effects ofototoxicity on both peripheral auditory and vestibular systems (Sera etal., 1987; Hinojosa and Lerner, 1987; Bareggi et al., 1990). Although itis generally believed that gentamicin destroys hair cells in all innerear structures (Warchol et al., 1993; Lefebvre et al., 1993; Duckert andRubel 1994), damage in the eighth nerve and cochleovestibular ganglionhas also been observed (Sera et al., 1987; Hinojosa and Lerner, 1987).In the present experiments, gentamicin induced VGN cell death wasobserved at concentrations of 1-3 mg/ml, which appears to be somewhathigher than the concentrations (0.5-1 mg/ml) needed to destroy haircells (Warchol et al., 1993; Lefebvre et al., 1993). Aminoglycosides athigh concentration may directly damage VGNs in vivo (Hinojosa andLerner, 1987).

[0198] It is interesting to note that VGNs are derived duringneurogenesis from the same neurogenic placode as the primary auditoryneurons in the spiral ganglia Both auditory neurons and VGNs sendperipheral projections to hair cells of the inner ear and extend centralprojections to the brain stem. Spiral ganglion neurons are alsoprotected from ototoxins by neurotrophins. While the central targetseems to have little effect on the survival of the two neuronal types,the peripheral target promotes their survival (Ard et al., 1985).Neurotrophins protect VGNs from deleterious effects of cisplatin, but inaddition, protect VGNs from gentamicin neurotoxicity. Although bothtypes of neurons show similar responses to the neurotrophins and producethe same types of receptors for neurotrophins, there are noticeabledifferences in terms of efficacy of the three neurotrophins, which mayreflect the ratio and level of neurotrophins available in the peripheraltarget and expression pattern of neurotrophin receptors in the two typesof neurons. For example, NT-4/5 and BDNF promote the survival of spiralganglion neurons up to 3-fold whereas only a 2-fold increase in thenumber of surviving VGNs is seen. In BDNF knockout mice, a majority ofVGNs are lost, while primary auditory innervation remains unaffected(Ernfors et al., 1994). Similarly, when the NT-3 gene is deleted, thedestruction of neurons in spiral ganglia is much more severe than in thevestibular ganglia (Farinas et al., 1994).

[0199] The results presented with organotypic cochlear explants areconsistent with the dissociated neuronal culture findings. As theorganotypic culture keeps the pertinent innervation of hair cellsintact, it better represents the in vivo system and, consequently,allows exploration of the mechanism of actions of ototoxins and, mostimportantly, provides a system to discover and test candidate protectiveagents.

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What is claimed is:
 1. A method for treating neuronal-related balanceimpairment in a mammal, comprising administering to a mammal in need ofsuch treatment a therapeutically effective amount of a trkB or trkCagonist.
 2. The method of claim 1, wherein the agonist is aneurotrophin.
 3. The method of claim 2, wherein the agonist is selectedfrom the group consisting of NT-4/5, BDNF or NT-3.
 4. The method ofclaim 3, wherein the agonist is NT-4/5
 5. The method of claim 1, whereinthe balance impairment is ototoxin induced.
 6. The method of claim 5,wherein the ototoxicity affects vestibular ganglion neurons.
 7. Themethod of claim 6, wherein the ototoxicity results from administrationof a therapeutically effective amount of an ototoxic compound selectedfrom the group consisting of a chemotherapeutic agent and an antibiotic.8. The method of claim 7, wherein the antibiotic is an aminoglycosideantibiotic.
 9. The method of claim 8, wherein the aminoglycosideantibiotic is selected from the group consisting of neomycin,paromomycin, ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,viomycin, gentamicin, sisomicin, netilmicin, streptomycin, dibekacin,fortimicin, and dihydrostreptomycin.
 10. The method of claim 7, whereinthe ototoxic compound is a chemotherapeutic agent.
 11. The method ofclaim 10, wherein the ototoxic compound is cisplatin or cisplatin-likecompound.
 12. The method of claim 7, wherein the trkB or trkC agonist isadministered prior to administration of an ototoxin.
 13. The method ofclaim 1, wherein the trkB or trkC agonist is administered with an agentthat promotes hair cell growth or regeneration.
 14. The method of claim1, which further comprises administering an effective amount of a secondtrkB or trkC agonist.
 15. The method of claim 1, wherein the agonistcomprises a chimeric or pantropic neurotrophin.
 16. The method of claim15, wherein the pantropic neurotrophin is MNTS-1.
 17. A method ofassaying for a trkB or trkC agonist that provides vestibular ganglionneuron protection or survival from an ototoxin, comprising, culturing autricle explant, administering a trkB or trkC agonist to the culture,administering an ototoxin to the culture, and determining the amount ofprotection or survival compared to a control culture to which the trkBor trkC agonist was not administered.
 18. A pharmaceutical composition,comprising a pharmaceutical agent capable of ototoxin-induced balanceimpairment and a trkB or trkC agonist in an amount therapeuticallyeffective for treating ototoxicity caused by the pharmaceutical agent.19. The pharmaceutical composition of claim 18, wherein the ototoxicpharmaceutical agent is selected from the group consisting of anaminoglycoside antibiotic or an antineoplastic agent.
 20. Thepharmaceutical composition of claim 18, further comprising a hair cellgrowth factor.
 21. An improved method for treatment of infection of amammal by administration of an aminoglycoside antibiotic, theimprovement comprising administering a therapeutically effective amountof a trkB or trkC agonist to the patient in need of such treatment toreduce or prevent ototoxin-induced balance impairment associated withthe antibiotic.
 22. An improved method for treatment of cancer in amammal by administration of a chemotherapeutic compound, the improvementcomprises administering a therapeutically effective amount of a trkB ortrkC agonist to the patient in need of such treatment to reduce orprevent ototoxin-induced balance impairment associated with thechemotherapeutic drug.
 23. A method for promoting vestibular ganglionneuron survival prior to, upon, or after exposure to an ototoxin or aninjury causing neuronal damage, loss, or degeneration, comprisingadministering to the neuron an effective amount of trkB or trkC agonist.